Methods and compositions for controlling efficacy of rna silencing

ABSTRACT

Based at least in part on an understanding of the mechanisms by which small RNAs (e.g., naturally-occurring miRNAs) mediate RNA silencing in plants, rules have been established for determining, for example, the degree of complementarity required between an RNAi-mediating agent and its target, i.e., whether mismatches are tolerated, the number of mismatches tolerated, the effect of the position of the mismatches, etc. Such rules are useful, in particular, in the design of improved RNAi-mediating agents which allow for more exact control of the efficacy of RNA silencing.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/917,480, filed Jun. 13, 2013, which is a continuation of U.S. patentapplication Ser. No. 12/802,534, filed Jun. 7, 2010, which is acontinuation of U.S. patent application Ser. No. 12/139,072, filed Jun.13, 2008 (now U.S. Pat. No. 7,732,593), which is a continuation of U.S.patent application Ser. No. 10/859,337, filed Jun. 2, 2004 (now U.S.Pat. No. 7,459,547), which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/475,386, filed Jun. 2, 2003.

U.S. patent application Ser. No. 13/917,480 is also a continuation ofU.S. patent application Ser. No. 12/802,536 filed Jun. 7, 2010, which isa divisional of Ser. No. 12/172,938, filed Jul. 14, 2008 (now U.S. Pat.No. 7,772,203), which is a divisional of U.S. patent application Ser.No. 10/859,337, filed Jun. 2, 2004 (now U.S. Pat. No. 7,459,547), whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/475,386, filed Jun. 2, 2003.

The entire disclosures of each of these patents and patent applicationsare incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.GM062862, GM065236, and GM05880 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) in animals and basal eukaryotes, quelling infungi, and posttranscriptional gene silencing (PTGS) in plants areexamples of a broad family of phenomena collectively called RNAsilencing (Kooter et al. 1999; Li and Ding 2001; Matzke et al. 2001;Vaucheret et al. 2001; Waterhouse et al. 2001; Hannon 2002; Plasterk2002). The unifying features of RNA silencing phenomena are theproduction of small (21-26 nt) RNAs that act as specificity determinantsfor down-regulating gene expression (Hamilton and Baulcombe 1999;Hammond et al. 2000; Parrish et al. 2000; Zamore et al. 2000; Djikeng etal. 2001; Parrish and Fire 2001; Tijsterman et al. 2002) and therequirement for one or more members of the Argonaute family of proteins(or PPD proteins, named for their characteristic PAZ and Piwi domains)(Tabara et al. 1999; Fagard et al. 2000; Hammond et al. 2001; Hutvagnerand Zamore 2002; Kennerdell et al. 2002; Martinez et al. 2002a;Pal-Bhadra et al. 2002; Williams and Rubin 2002).

Small RNAs are generated in animals by members of the Dicer family ofdouble-stranded RNA (dsRNA)-specific endonucleases (Bernstein et al.2001; Billy et al. 2001; Grishok et al. 2001; Ketting et al. 2001).Dicer family members are large, multidomain proteins that containputative RNA helicase, PAZ, two tandem ribonuclease III (RNase III), andone or two dsRNA-binding domains. The tandem RNase III domains arebelieved to mediate endonucleolytic cleavage of dsRNA into smallinterfering RNAs (siRNAs), the mediators of RNAi. In Drosophila andmammals, siRNAs, together with one or more Argonaute proteins, form aprotein-RNA complex, the RNA-induced silencing complex (RISC), whichmediates the cleavage of target RNAs at sequences with extensivecomplementarity to the siRNA (Hammond et al. 2000, 2001; Zamore et al.2000; Elbashir et al. 2001a,b,c; Nykänen et al. 2001; Hutvagner andZamore 2002; Martinez et al. 2002a).

In addition to Dicer and Argonaute proteins, RNA-dependent RNApolymerase (RdRP) genes are required for RNA silencing in Caenorhabditiselegans (Smardon et al. 2000; Sijen et al. 2001), Neurospora crassa(Cogoni and Macino 1999), and Dictyostelium discoideum (Martens et al.2002), but likely not for RNAi in Drosophila or mammals (Celotto andGraveley 2002; Chiu and Rana 2002; Holen et al. 2002; Martinez et al.2002b; Schwarz et al. 2002; Roignant et al. 2003). In plants, PTGSinitiated by transgenes that overexpress an endogenous mRNA alsorequires a putative RdRP, SGS2 (SDE1; Dalmay et al. 2000; Mourrain etal. 2000), although transgenes designed to generate dsRNA bypass thisrequirement (Beclin et al. 2002). Similarly, silencing induced byviruses replicating through a dsRNA intermediate (virus-induced genesilencing, VIGS) does not require SGS2 (Dalmay et al. 2000).

Dicer in animals and CARPEL FACTORY (CAF, a Dicer homolog) in plantsalso generate microRNAs (miRNAs), 20-24-nt, single-stranded noncodingRNAs thought to regulate endogenous mRNA expression (Lee et al. 1993;Reinhart et al. 2000, 2002; Grishok et al. 2001; Hutvagner et al. 2001;Ketting et al. 2001; Lagos-Quintana et al. 2001, 2002; Lau et al. 2001;Lee and Ambros 2001; Mourelatos et al. 2002; Park et al. 2002). miRNAsare produced by Dicer cleavage of stem-loop precursor RNA transcripts(pre-miRNAs); the miRNA can reside on either the 5′ or 3′ side of thedouble-stranded stem (Lee et al. 1993; Pasquinelli et al. 2000;Lagos-Quintana et al. 2001; Lau et al. 2001; Lee and Ambros 2001). Inanimals, pre-miRNAs are transcribed as longer primary transcripts(pri-miRNAs) that are processed in the nucleus into compact, foldedstructures (pre-miRNAs), then exported to the cytoplasm, where they arecleaved by Dicer to yield mature miRNAs (Lee et al. 2002). Animal miRNAsare only partially complementary to their target mRNAs; this partialcomplementarity has been proposed to cause miRNAs to repress translationof their targets, rather than direct target cleavage by the RNAi pathway(for review, see Ruvkun 2001; Hutvágner and Zamore 2002). Plant miRNAshave far greater complementarity to cellular mRNAs and have beenproposed to mediate target RNA cleavage via an RNAi-like mechanism(Llave et al. 2002b; Rhoades et al. 2002).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the finding thatextracts of wheat germ recapitulate many of the key features of RNAsilencing in plants. Using this in vitro system, it is shown that inplants, ATP-dependent, Dicer-like enzymes cleave dsRNA into small RNAsthat have the structure of siRNAs. Unlike Drosophila embryos ormammalian cells, plants convert dsRNA into two distinct classes ofsiRNAs, long and short siRNAs. Inhibitor studies indicate that adifferent Dicer-like enzyme generates each siRNA class. Furthermore, awheat RdRP activity can synthesize dsRNA using exogenous single-strandedRNA as a template without an exogenous primer, and that this dsRNA ispreferentially converted into long siRNAs.

Wheat germ extracts also contain an endogenous RISC programmed with amiRNA which can direct efficient cleavage of the wild-type ArabidopsisPHAVOLUTA (PHV) mRNA sequence, but not that of a previously describeddominant PHV mutant. Interestingly, exact complementarity between themiRNA and target mRNA is not necessary for the miRNA to direct efficienttarget cleavage. An siRNA containing three mismatches with its targetmRNA, was found to be at least as potent as an siRNA with perfectcomplementarity to the same target sequence, demonstrating thatmismatches per se do not block target cleavage. Rather, the specificposition and sequence of siRNA:target RNA mismatches determine if theypermit or disrupt RNAi. It is proposed that three or four mismatchesbetween an miRNA (or the guide strand of an siRNA duplex) and its targetRNA, properly placed so as to still permit mRNA cleavage, facilitatesthe release of cleaved target RNA from the RISC complex, therebyincreasing the rate of enzyme turnover. In particular, the efficiency ofcleavage is greater when a G:U base pair, referred to also as a G:Uwobble, is present near the 5′ or 3′ end of the complex formed betweenthe miRNA and the target. Understanding the natural mechanism by whichmiRNAs efficiency mediate RNAi in plants allows for the design ofimproved RNAi agents for use in mediating RNAi not only in plants, butin eukaryotes (in particular, in mammals).

Accordingly, the present invention features methods of enhancing theefficacy of an RNAi agent comprising substituting a at least oneterminal nucleotide with a nucleotide that does not form a Watson-Crickbase pair with the corresponding nucleotide in a target mRNA. Theinvention also provides compositions comprising RNAi agents, e.g.,siRNAs, pre-miRNA, shRNAs, having nucleotide substitutions for enhancedefficacy of RNAi, as well as vectors, transgenes and cells comprisingthe RNAi agents. Further featured is a Dicer-like enzyme, an extractcomprising the enzyme, and methods for their use. Kits for use inmediating RNAi comprising the compositions of the invention areprovided. Therapeutic methods and pharmaceutical compositions are alsoprovided.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Arabidopsis thaliana small RNAs form two distinct sizeclasses. (FIG. 1A) Size distribution of small RNA clones. (FIG. 1B)Sequence composition of the 5′ ends of cloned small RNA as a function oflength.

FIGS. 2A-2G. dsRNA is cleaved into two discrete classes of bona fidesiRNAs in plant extracts. (FIG. 2A) Upon incubation in wheat germextract, 32P-dsRNA was cleaved into small RNAs in a highly processivereaction, as in fly embryo lysate. (FIG. 2B) 32P-dsRNA was cleaved inwheat germ extract into two sizes of small RNAs, ˜21-nt and 24-25-ntlong, relative to synthetic 5′-32P-radiolabeled RNA markers. (FIG. 2C)32P-dsRNA was cleaved in cauliflower extract into two sizes of smallRNAs. (FIG. 2D) Efficient production of small RNAs in wheat germ extractrequired ATP. ATP, creatine phosphate, and creatine kinase were included(+ATP) or omitted ([−] ATP) from the reaction. (FIG. 2E) Small RNAsproduced in vitro in wheat germ extract are double-stranded. 32P-dsRNAwas incubated in wheat germ extract or Drosophila embryo lysate,deproteinized at room temperature without organic extraction, thenanalyzed by gel filtration on a Superdex 200 HR column. The peakpositions of double- and single-stranded synthetic siRNA standards areindicated. (FIG. 2F) Scheme for detecting 3′ overhanging ends on smallRNAs by nuclease protection. (FIG. 2G) Small RNAs produced by incubationof 32P-dsRNA in wheat germ extract have ˜2-nt 3′ overhanging ends and acentral double-stranded body, characteristics of the products of Dicercleavage. Brackets indicate the nuclease digestion products. Thepositions of 5′-32P-radiolabeled size markers are indicated at left.3′-phosphorylated markers were generated by reacting synthetic RNAs onebase longer than indicated with periodate, followed by[beta]-elimination, yielding an RNA one base shorter, but bearing a3′-phosphate in place of a hydroxyl.

FIGS. 3A-3C. The two classes of plant siRNAs are produced by differentenzymes. (FIG. 3A, FIG. 3B) 32P-dsRNA was incubated in either Drosophilaembryo lysate or wheat germ extract for 3 hours in the presence ofincreasing concentrations of 21-nt or 25-nt siRNA duplexes, thenanalyzed by denaturing gel electrophoresis and quantified. The siRNAconcentration is presented in micromoles nucleotide per liter to permitcomparison of the different lengths of siRNA duplex used. The relativeefficiency of the reactions was determined by fitting the data to asingle exponential and comparing the rate constant. (FIG. 3A) 21-ntsiRNA duplexes (filled circles) are more efficient inhibitors ofDrosophila Dicer than 25-nt siRNA duplexes (open circles). (FIG. 3B)Production of 25-nt siRNAs in wheat germ extract (squares) was inhibitedmore efficiently by a 25-nt synthetic siRNA duplex (red symbols) than bya 21-nt siRNA duplex (black symbols), but production of the 21-nt siRNAs(circles) was not inhibited by either synthetic siRNA duplex. (FIG. 3C)dsRNA competitor inhibited the production of both 25-nt (black squares)and 21-nt (red circles) siRNAs in wheat germ extract. Production ofsiRNAs in Drosophila embryo lysate (blue circles) was also inhibited bydsRNA competitor, but to a lower extent, perhaps reflecting a higherconcentration of Dicer in Drosophila embryo lysate than in wheat germextract.

FIG. 4. Wheat germ extract contains an RdRP activity. Single-strandedRNA of the indicated size and cap structure was incubated in wheat germextract for 3 hours in the presence of ATP, CTP, GTP, and α-32P-UTP. Theproducts of the reaction were analyzed by denaturing polyacrylamide gelelectrophoresis.

FIGS. 5A-5C. Characterization of the wheat RdRP activity. (FIG. 5A)Wheat germ extract, but not Drosophila embryo lysate, contains an RdRPactivity that can extend a primer. The arrowhead indicates the primerextension product produced when an antisense 21-nt RNA primer, but not asense primer, was incubated in the wheat germ extract with a 592-ntsingle-stranded RNA. The primers correspond to nucleotides 511-532 ofthe RNA template. (FIG. 5B) RdRP-dependent production of small RNAs inwheat germ extract. Increasing concentrations of a 2.7-kb Photinuspyralis (Pp) luciferase mRNA triggered production of 32P-radiolabeledsmall RNAs in wheat germ extract when ribonucleotide triphosphates(including α-32P-UTP), but not when 3′-deoxy GTP and 3′-deoxy CTP wereincluded in the reaction. (FIG. 5C) Production of newly synthesizedsmall RNAs was more efficiently inhibited by a 25-nt synthetic siRNAduplex (open circles) than by a 21-nt synthetic siRNA duplex (opensquares).

FIGS. 6A-6C. miR165/166 in wheat germ extract. (FIG. 6A) A wheatortholog of miR165 or miR166 is present in wheat germ extract.Quantitative Northern hybridization analysis using synthetic miR165 RNAconcentration standards, antisense miR165 RNA, and total RNA preparedfrom 30 μL of wheat germ extract or Drosophila embryo lysate. (FIG. 6B)Quantitation of the data in A. Closed circles, synthetic miR165standards; open circle, RNA extracted from 30 μL of wheat germ extract.The line shows a linear fit of the four highest concentration standards.(FIG. 6C) Schematic of the RNA targets, indicating the sequences of themiR165/166-complementary regions of wild-type PHV and mutant phv mRNAs,miR165, miR166, and the siRNA antisense strands used in FIG. 7C.

FIGS. 7A-7C. An endogenous wheat nuclease efficiently cleaves wild-typebut not mutant PHV target RNAs. (FIG. 7A) When incubated in wheat germextract, 5′-radiolabeled target RNA containing wild-type PHV sequenceswas cleaved within the PHV sequences complementary to miR165 and miR166.In contrast, a dominant G→A mutant target RNA was cleaved inefficiently.(FIG. 7B) Quantification of the data in A. (Circles) Wild-type PHVsequences; (squares) mutant sequences; (filled symbols) full-lengthtarget RNA; (open symbols) 5′ cleavage product. The difference incleavage rates is ˜14-fold. (FIG. 7C) Analysis of PHV cleavage in an invitro RNAi reaction programmed with siRNA duplexes and Drosophila embryolysate. The identity of the antisense strand of the siRNA duplex and theRNA target used is indicated above the gel and described in FIG. 6C.

FIG. 8. Quantification of the fraction of target mRNA cleaved by siRNAhaving perfect complementarity to target versus siRNA having twomismatched with target (miR 165 siRNA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatextracts of wheat germ, introduced for the study of translation andprotein translocation in the 1970s (Roberts and Paterson 1973),recapitulate many of the key features of RNA silencing in plants. Usingthis in vitro system, the instant inventors have shown that in plants,ATP-dependent, Dicer-like enzymes cleave dsRNA into small RNAs that havethe structure of siRNAs. Unlike Drosophila embryos or mammalian cells,plants convert dsRNA into two distinct classes of siRNAs, long (e.g.,21-22 nucleotides) and short (e.g., 24-25 nucleotides) siRNAs. Inhibitorstudies indicate that a second Dicer-like enzyme functions in plants togenerate each siRNA class. The instant inventors have also shown that awheat RdRP activity can synthesize dsRNA using exogenous single-strandedRNA as a template without an exogenous primer, and that this dsRNA ispreferentially converted into long siRNAs.

Finally, it is demonstrated that wheat germ extracts contain anendogenous RISC programmed with a miRNA. This endogenous miRNA complexhas sufficient sequence information to direct efficient cleavage of thewild-type Arabidopsis PHA VOLUTA (PHV) mRNA sequence, but not that of apreviously described dominant PHV mutant that perturbs leaf development.Based on an understanding of the mechanism by which miRNAs direct RNAiin plants, new siRNAs can be designed for regulating RNAi in plants.More importantly, siRNAs can be designed, for example, based on thesequence of various eukaryotic miRNAs, such siRNAs having utility inmediating RNAi in mammals, and particularly, in humans.

Accordingly, in one aspect, the instant invention provides a method ofenhancing the efficacy of an RNAi agent, involving substituting at leastone terminal nucleotide of the RNAi agent with a nucleotide which doesnot form a Watson-Crick base pair with the corresponding nucleotide in atarget mRNA, such that efficacy is enhanced.

In one embodiment, the substituted nucleotide forms a G:U wobble basepair with the target mRNA. In one preferred embodiment, the substitutionis an A→G substitution, the G forming a G:U wobble base pair with a U inthe corresponding target mRNA. In another preferred embodiment, thesubstitution is a C→U substitution, the U forming a G:U wobble base pairwith a G in the corresponding target mRNA.

In one embodiment, the terminal nucleotide is within 5 or fewernucleotides from the 5′ end of the RNAi agent. In a related embodiment,the terminal nucleotide is within 5 or fewer nucleotides from the 3′ endof the RNAi agent.

In one embodiment, at least two terminal nucleotides are substituted. Inpreferred embodiments, the two terminal nucleotides substituted are atthe 5′ end of the RNAi agent or at the 3′ end of the RNAi agent. Inanother preferred embodiment, a first terminal nucleotide substituted isat the 5′ end of the RNAi agent and a second terminal nucleotidesubstituted is at the 3′ end of the RNAi agent.

In one embodiment, at least three, four or five terminal nucleotides aresubstituted.

In another aspect, the instant invention provides a RNAi agent having atleast one terminal nucleotide of the RNAi agent substituted with anucleotide which forms a G:U wobble base pair with the correspondingnucleotide in a target mRNA.

In one embodiment of this aspect of the invention, the substitution isan A→G substitution, the G forming a G:U wobble base pair with a U inthe corresponding target mRNA. In another embodiment, the substitutionis a C→U substitution, the U forming a G:U wobble base pair with a G inthe corresponding target mRNA.

In other embodiments, the terminal nucleotide is within 5 or fewernucleotides from the 5′ end of the RNAi agent or from the 3′ end of theRNAi agent.

In yet other embodiments, at least two terminal nucleotides aresubstituted. In preferred embodiments, the two terminal nucleotidessubstituted are at the 5′ end of the RNAi agent or at the 3′ end of theRNAi agent. In other preferred embodiments, a first terminal nucleotidesubstituted is at the 5′ end of the RNAi agent and a second terminalnucleotide substituted is at the 3′ end of the RNAi agent.

In other embodiments, at least three, four or five terminal nucleotidesare substituted.

In various embodiments of this aspect of the invention, the RNAi agentis chemically synthesized, enzymatically synthesized, or derived from anengineered precursor.

In another aspect, the instant invention provides a method of enhancingsilencing of a target mRNA, comprising contacting a cell having an RNAipathway with the RNAi agent of any one of the preceding claims underconditions such that silencing is enhanced.

In yet another aspect, the instant invention provides a method ofenhancing silencing of a target mRNA in a subject, comprisingadministering to the subject a pharmaceutical composition comprising theRNAi agent of any one of the preceding claims such that silencing isenhanced.

In certain embodiments of the invention, compositions are providedcomprising the RNAi agents of the invention formulated to facilitateentry of the agent into a cell. Pharmaceutical compositions comprisingthe RNAi agents of the invention are also provided.

In other embodiments, the instant invention provides engineeredpre-miRNA comprising the RNAi agent of any one of the preceding claims,and vectors encoding the pre-miRNA.

In related embodiments, the instant invention provides a pri-miRNAcomprising the pre-miRNA of the invention, and a vector encoding thepri-miRNA.

In yet other embodiments, the invention provides a small hairpin RNA(shRNA) comprising nucleotide sequence identical to any of the RNAiagents of the instant invention, a vector encoding the shRNA, and atransgene encoding the shRNA.

The instant invention further provides a cell, e.g., a mammalian cell,preferably a human cell, comprising the vectors of the invention. 35.

In another aspect, the instant invention provides an isolatedArabidopsis thaliana Dicer-like enzyme capable of cleaving a long dsRNAsubstrate into short, 24-25 nucleotide dsRNA products, the activity ofsaid enzyme being inhibited in the presence of said dsRNA products. In arelated aspect, the instant invention provides a method of generating aRNAi agent 24-25 nucleotides in length, comprising incubating a dsRNAsubstrate with the enzyme of the invention, such that the agent isgenerated. Also provided is an Arabidopsis thaliana cell-free extractcomprising the enzyme of the invention. In a related aspect, a method isprovided of generating a RNAi agent 24-25 nucleotides in length,comprising incubating a dsRNA substrate with the extract of theinvention, such that the agent is generated.

In a certain embodiment of the instant invention, a kit is provided foruse in mediating RNAi, comprising the enzyme or the extract of theinvention, and instructions for use.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. Additional exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine,²N-methylguanosine and ^(2,2)N,N-dimethylguanosine (also referred to as“rare” nucleosides). The term “nucleotide” refers to a nucleoside havingone or more phosphate groups joined in ester linkages to the sugarmoiety. Exemplary nucleotides include nucleoside monophosphates,diphosphates and triphosphates. The terms “polynucleotide” and “nucleicacid molecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides. The term “DNA” or “DNA molecule” ordeoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.Preferably, an siRNA comprises between about 15-30 nucleotides ornucleotide analogs, more preferably between about 16-25 nucleotides (ornucleotide analogs), even more preferably between about 18-23nucleotides (or nucleotide analogs), and even more preferably betweenabout 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22nucleotides or nucleotide analogs). The term “short” siRNA refers to asiRNA comprising ˜21 nucleotides (or nucleotide analogs), for example,19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNAcomprising ˜24-25 nucleotides, for example, 23, 24, 25 or 26nucleotides. Short siRNAs may, in some instances, include fewer than 19nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shortersiRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, insome instances, include more than 26 nucleotides, provided that thelonger siRNA retains the ability to mediate RNAi absent furtherprocessing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Preferred nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivitized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino) propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; 0- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phophoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

A RNAi agent having a strand which is “sequence sufficientlycomplementary to a target mRNA sequence to direct target-specific RNAinterference (RNAi)” means that the strand has a sequence sufficient totrigger the destruction of the target mRNA by the RNAi machinery orprocess.

The term “phosphorylated” means that at least one phosphate group isattached to a chemical (e.g., organic) compound. Phosphate groups can beattached, for example, to proteins or to sugar moieties via thefollowing reaction: free hydroxyl group+phosphate donor→phosphate esterlinkage. The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the 5′ sugar (e.g., the 5′ribose or deoxyribose, or an analog of same). Mono-, di-, andtriphosphates are common. Also intended to be included within the scopeof the instant invention are phosphate group analogs which function inthe same or similar manner as the mono-, di-, or triphosphate groupsfound in nature (see e.g., exemplified analogs.)

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or“isolated siRNA precursor”) refers to RNA molecules which aresubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

As used herein, the term “transgene” refers to any nucleic acidmolecule, which is inserted by artifice into a cell, and becomes part ofthe genome of the organism that develops from the cell. Such a transgenemay include a gene that is partly or entirely heterologous (i.e.,foreign) to the transgenic organism, or may represent a gene homologousto an endogenous gene of the organism. The term “transgene” also means anucleic acid molecule that includes one or more selected nucleic acidsequences, e.g., DNAs, that encode one or more engineered RNAprecursors, to be expressed in a transgenic organism, e.g., animal,which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal, or homologous to an endogenous gene of the transgenicanimal, but which is designed to be inserted into the animal's genome ata location which differs from that of the natural gene. A transgeneincludes one or more promoters and any other DNA, such as introns,necessary for expression of the selected micleic acid sequence, alloperably linked to the selected sequence, and may include an enhancersequence.

A gene “involved” in a disorder includes a gene, the normal or aberrantexpression or function of which effects or causes a disease or disorderor at least one symptom of said disease or disorder

The phrase “examining the function of a gene in a cell or organism”refers to examining or studying the expression, activity, function orphenotype arising therefrom.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc., to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc., determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc., can be determinedprior to introducing a RNAi agent of the invention into a cell ororganism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. RNA Molecules

The present invention features “RNAi agents”, methods of making saidRNAi agents and methods (e.g., research and/or therapeutic methods) forusing said RNAi agents. The RNAi agents can be siRNA molecules,precursor molecules (e.g., engineered precursor molecules) that areprocessed into siRNA molecules, or molecules (e.g., DNA molecules) thatencode, for example, precursor molecules (e.g., engineered precursormolecules)

Exemplary siRNA molecules have a length from about 10-50 or morenucleotides. Preferably, siRNA molecule has a length from about 15-45 or15-30 nucleotides. More preferably, the siRNA molecule has a length fromabout 16-25 or 18-23 nucleotides. The siRNA molecules of the inventionfurther comprise at least one strand that has a sequence that is“sufficiently complementary” to a target mRNA sequence to directtarget-specific RNA interference (RNAi), as defined herein, i.e., thestrand has a sequence sufficient to trigger the destruction of thetarget mRNA by the RNAi machinery or process. Such a strand can bereferred to as an antisense strand in the context of a ds-siRNAmolecule. The siRNA molecule can be designed such that every residue iscomplementary to a residue in the target molecule. Preferably, however,the siRNA molecule is designed such that modified base pairing, inparticular, G:U base pairing (i.e., G:U “wobble” base pairing) occursbetween the strand of the siRNA molecule mediating RNAi and the targetmRNA.

In further embodiments, substitutions can be made within the molecule toincrease stability and/or enhance processing activity of said molecule.Substitutions can be made within the strand or can be made to residues athe ends of the strand. Preferably, however, substitutions are not madein the central portion of the strand as the sequence of this portion ofthe strand has been determined to be essential to effecting cleavage ofthe corresponding target mRNA. The 5′-terminus is, most preferably,phosphorylated (i.e., comprises a phosphate, diphosphate, ortriphosphate group). The 3′ end of an siRNA can be a hydroxyl groupalthough there is no requirement for a 3′ hydroxyl group when the activeagent is a ss-siRNA molecule.

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNA containing a nucleotide sequences identicalto a portion of the target gene are preferred for inhibition. However,100% sequence identity between the siRNA and the target gene is notrequired to practice the present invention. Thus the invention has theadvantage of being able to tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence. For example, siRNA sequences with insertions, deletions, andsingle point mutations relative to the target sequence have also beenfound to be effective for inhibition. Alternatively, siRNA sequenceswith nucleotide analog substitutions or insertions can be effective forinhibition.

Moreover, not all positions of a siRNA contribute equally to targetrecognition. Mismatches in the center of the siRNA are most critical andessentially abolish target RNA cleavage. In contrast, the 3′ nucleotidesof the siRNA do not contribute significantly to specificity of thetarget recognition. In particular, residues 3′ of the siRNA sequencewhich is complementary to the target RNA (e.g., the guide sequence) arenot critical for target RNA cleavage.

Sequence identity may determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between a strand of theRNAi agent and the portion of the target gene is preferred.Alternatively, the RNAi agent may be defined functionally as anucleotide sequence (or oligonucleotide sequence) that is capable ofhybridizing with a portion of the target gene transcript (e.g., 400 mMNaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for12-16 hours; followed by washing). Additional preferred hybridizationconditions include hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC,50% formamide followed by washing at 70° C. in 0.3×SSC or hybridizationat 70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washingat 67° C. in 1×SSC. The hybridization temperature for hybridsanticipated to be less than 50 base pairs in length should be 5-10° C.less than the melting temperature (Tm) of the hybrid, where Tm isdetermined according to the following equations. For hybrids less than18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases).For hybrids between 18 and 49 base pairs in length, Tm(°C.)=81.5+16.6(log 10[Na+])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer ([Na+] for 1×SSC=0.165 M). Additional examplesof stringency conditions for polynucleotide hybridization are providedin Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., chapters 9 and 11, and Current Protocols inMolecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons,Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. Thelength of the identical nucleotide sequences may be at least about 10,12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

In a preferred aspect, the RNA molecules of the present invention aremodified to improve stability in serum or in growth medium for cellcultures. In order to enhance the stability, the 3′-residues may bestabilized against degradation, e.g., they may be selected such thatthey consist of purine nucleotides, particularly adenosine or guanosinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference. Forexample, the absence of a 2′ hydroxyl may significantly enhance thenuclease resistance of the RNA agents in tissue culture medium.

In an especially preferred embodiment of the present invention the RNAmolecule may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific activity, e.g., the RNAi mediating activity is notsubstantially effected, e.g., in a region at the 5′-end and/or the3′-end of the RNA molecule. Particularly, the ends may be stabilized byincorporating modified nucleotide analogues.

Preferred nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; 0-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

RNA may be produced enzymatically or by partial/total organic synthesis,any modified nibonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, a RNAi agent is preparedchemically. Methods of synthesizing RNA molecules are known in the art,in particular, the chemical synthesis methods as de scribed in Verma andEckstein (1998) Annul Rev. Biochem. 67:99-134. In another embodiment, aRNAi agent is prepared enzymatically. For example, a ds-siRNA can beprepared by enzymatic processing of a long ds RNA having sufficientcomplementarity to the desired target mRNA. Processing of long ds RNAcan be accomplished in vitro, for example, using appropriate cellularlysates and ds-siRNAs can be subsequently purified by gelelectrophoresis or gel filtration. ds-siRNA can then be denaturedaccording to art-recognized methodologies. In an exemplary embodiment,RNA can be purified from a mixture by extraction with a solvent orresin, precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the RNA may be used with no or a minimum ofpurification to avoid losses due to sample processing. Alternatively,the single-stranded RNAs can also be prepared by enzymatic transcriptionfrom synthetic DNA templates or from DNA plasmids isolated fromrecombinant bacteria. Typically, phage RNA polymerases are used such asT7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck (1989) MethodsEnzymol. 180:51-62). The RNA may be dried for storage or dissolved in anaqueous solution. The solution may contain buffers or salts to inhibitannealing, and/or promote stabilization of the single strands.

In one embodiment, the target mRNA of the invention specifies the aminoacid sequence of a cellular protein (e.g., a nuclear, cytoplasmic,transmembrane, or membrane-associated protein). In another embodiment,the target mRNA of the invention specifies the amino acid sequence of anextracellular protein (e.g., an extracellular matrix protein or secretedprotein). As used herein, the phrase “specifies the amino acid sequence”of a protein means that the mRNA sequence is translated into the aminoacid sequence according to the rules of the genetic code. The followingclasses of proteins are listed for illustrative purposes: developmentalproteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN,NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressorproteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53,and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturasesand hydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression of the host, replication of the pathogen, transmissionof the pathogen, or maintenance of the infection), or a host proteinwhich facilitates entry of the pathogen into the host, drug metabolismby the pathogen or host, replication or integration of the pathogen'sgenome, establishment or spread of infection in the host, or assembly ofthe next generation of pathogen. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

In one embodiment, the target mRNA molecule of the invention specifiesthe amino acid sequence of an endogenous protein (i.e., a proteinpresent in the genome of a cell or organism). In another embodiment, thetarget mRNA molecule of the invention specified the amino acid sequenceof a heterologous protein expressed in a recombinant cell or agenetically altered organism. In another embodiment, the target mRNAmolecule of the invention specified the amino acid sequence of a proteinencoded by a transgene (i.e., a gene construct inserted at an ectopicsite in the genome of the cell). In yet another embodiment, the targetmRNA molecule of the invention specifies the amino acid sequence of aprotein encoded by a pathogen genome which is capable of infecting acell or an organism from which the cell is derived.

By inhibiting the expression of such proteins, valuable informationregarding the function of said proteins and therapeutic benefits whichmay be obtained from said inhibition may be obtained.

In one embodiment, RNAi agents are synthesized either in vivo, in situ,or in vitro. Endogenous RNA polymerase of the cell may mediatetranscription in vivo or in situ, or cloned RNA polymerase can be usedfor transcription in vivo or in vitro. For transcription from atransgene in vivo or an expression construct, a regulatory region (e.g.,promoter, enhancer, silencer, splice donor and acceptor,polyadenylation) may be used to transcribe the RNAi agent. Inhibitionmay be targeted by specific transcription in an organ, tissue, or celltype; stimulation of an environmental condition (e.g., infection,stress, temperature, chemical inducers); and/or engineeringtranscription at a developmental stage or age. A transgenic organismthat expresses an RNAi agent from a recombinant construct may beproduced by introducing the construct into a zygote, an embryonic stemcell, or another multipotent cell derived from the appropriate organism.

II. Short hairpin RNAs (shRNAs)

In certain featured embodiments, the instant invention features shRNAswhich can be processed into siRNAs, for example, by a cell's endogenousRNAi machinery. In contrast to short siRNA duplexes, short hairpin RNAs(shRNAs) mimics the natural precursors of miRNAs and enters at the topof the RNAi pathway. For this reason, shRNAs are believed to mediateRNAi more efficiently by being fed through the entire natural RNAipathway.

1. Engineered RNA Precursors that Generate siRNAs

Naturally-occurring miRNA precursors (pre-miRNA) have a single strandthat forms a duplex stem including two portions that are generallycomplementary, and a loop, that connects the two portions of the stem.In typical pre-miRNAs, the stem includes one or more bulges, e.g., extranucleotides that create a single nucleotide “loop” in one portion of thestem, and/or one or more unpaired nucleotides that create a gap in thehybridization of the two portions of the stem to each other. Shorthairpin RNAs, or engineered RNA precursors, of the invention areartificial constructs based on these naturally occurring pre-miRNAs, butwhich are engineered to deliver desired siRNAs.

In shRNAs, or engineered precursor RNAs, of the instant invention, oneportion of the duplex stem is a nucleic acid sequence that iscomplementary (or anti-sense) to the target mRNA. Thus, engineered RNAprecursors include a duplex stem with two portions and a loop connectingthe two stem portions. The two stem portions are about 18 or 19 to about25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. When usedin mammalian cells, the length of the stem portions should be less thanabout 30 nucleotides to avoid provoking non-specific responses like theinterferon pathway. In non-mammalian cells, the stem can be longer than30 nucleotides. In fact, the stem can include much larger sectionscomplementary to the target mRNA (up to, and including the entire mRNA).The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 micleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us. The loop in the shRNAs orengineered RNA precursors may differ from natural pre-miRNA sequences bymodifying the loop sequence to increase or decrease the number of pairednucleotides, or replacing all or part of the loop sequence with atetraloop or other loop sequences. Thus, the loop in the shRNAs orengineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g.,15 or 20, or more nucleotides in length.

shRNAs of the invention include the sequences of the desired siRNAduplex. The desired siRNA duplex, and thus both of the two stem portionsin the engineered RNA precursor, are selected by methods known in theart. These include, but are not limited to, selecting an 18, 19, 20, 21nucleotide, or longer, sequence from the target gene mRNA sequence froma region 100 to 200 or 300 nucleotides on the 3′ side of the start oftranslation. In general, the sequence can be selected from any portionof the mRNA from the target gene, such as the 5′ UTR (untranslatedregion), coding sequence, or 3′ UTR. This sequence can optionally followimmediately after a region of the target gene containing two adjacent AAnucleotides. The last two nucleotides of the 21 or so nucleotidesequence can be selected to be UU (so that the anti-sense strand of thesiRNA begins with UU). This 21 or so nucleotide sequence is used tocreate one portion of a duplex stem in the engineered RNA precursor.This sequence can replace a stem portion of a wild-type pre-stRNAsequence, e.g., enzymatically, or is included in a complete sequencethat is synthesized. For example, one can synthesize DNAoligonucleotides that encode the entire stem-loop engineered RNAprecursor, or that encode just the portion to be inserted into theduplex stem of the precursor, and using restriction enzymes to build theengineered RNA precursor construct, e.g., from a wild-type pre-stRNA.

Engineered RNA precursors include in the duplex stem the 21-22 or sonucleotide sequences of the siRNA desired to be produced in vivo. Thus,the stem portion of the engineered RNA precursor includes at least 18 or19 nucleotide pairs corresponding to the sequence of an exonic portionof the gene whose expression is to be reduced or inhibited. The two 3′nucleotides flanking this region of the stem are chosen so as tomaximize the production of the siRNA from the engineered RNA precursor,and to maximize the efficacy of the resulting siRNA in targeting thecorresponding mRNA for destruction by RNAi in vivo and in vitro.

Another defining feature of these engineered RNA precursors is that as aconsequence of their length, sequence, and/or structure, they do notinduce sequence non-specific responses, such as induction of theinterferon response or apoptosis, or that they induce a lower level ofsuch sequence non-specific responses than long, double-stranded RNA(>150 bp) that has been used to induce RNAi. For example, the interferonresponse is triggered by dsRNA longer than 30 base pairs.

2. Transgenes Encoding Engineered RNA Precursors

The new engineered RNA precursors can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.).These synthetic, engineered RNA precursors can be used directly asdescribed below or cloned into expression vectors by methods known inthe field. The engineered RNA precursors should be delivered to cells invitro or in vivo in which it is desired to target a specific mRNA fordestruction. A number of methods have been developed for delivering DNAor RNA to cells. For example, for in vivo delivery, molecules can beinjected directly into a tissue site or administered systemically. Invitro delivery includes methods known in the art such as electroporationand lipofection.

To achieve intracellular concentrations of the nucleic acid moleculesufficient to suppress expression of endogenous mRNAs, one can use, forexample, a recombinant DNA construct in which the oligonucleotide isplaced under the control of a strong Pol III (e.g., U6 or Pol III H1-RNApromoter) or Pol II promoter. The use of such a construct to transfecttarget cells in vitro or in vivo will result in the transcription ofsufficient amounts of the engineered RNA precursor to lead to theproduction of an siRNA that can target a corresponding mRNA sequence forcleavage by RNAi to decrease the expression of the gene encoding thatmRNA. For example, a vector can be introduced in vivo such that it istaken up by a cell and directs the transcription of an engineered RNAprecursor. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredstRNA precursor.

Such vectors can be constructed by recombinant DNA technology methodsknown in the art. Vectors can be plasmid, viral, or other vectors knownin the art such as those described herein, used for replication andexpression in mammalian cells or other targeted cell types. The nucleicacid sequences encoding the engineered RNA precursors can be preparedusing known techniques. For example, two synthetic DNA oligonucleotidescan be synthesized to create a novel gene encoding the entire engineeredRNA precursor. The DNA oligonucleotides, which will pair, leavingappropriate ‘sticky ends’ for cloning, can be inserted into arestriction site in a plasmid that contains a promoter sequence (e.g., aPol II or a Pol III promoter) and appropriate terminator sequences 3′ tothe engineered RNA precursor sequences (e.g., a cleavage andpolyadenylation signal sequence from SV40 or a Pol III terminatorsequence).

The invention also encompasses genetically engineered host cells thatcontain any of the foregoing expression vectors and thereby express thenucleic acid molecules of the invention in the host cell. The host cellscan be cultured using known techniques and methods (see, e.g., Cultureof Animal Cells (R. I. Freshney, Alan R. Liss, Inc. 1987); MolecularCloning, Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989)).

Successful introduction of the vectors of the invention into host cellscan be monitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection canbe indicated using markers that provide the transfected cell withresistance to specific environmental factors (e.g., antibiotics anddrugs), such as hygromycin B resistance, e.g., in insect cells and inmammalian cells.

3. Regulatory Sequences

The expression of the engineered RNA precursors is driven by regulatorysequences, and the vectors of the invention can include any regulatorysequences known in the art to act in mammalian cells, e.g., human ormurine cells; in insect cells; in plant cells; or other cells. The termregulatory sequence includes promoters, enhancers, and other expressioncontrol elements. It will be appreciated that the appropriate regulatorysequence depends on such factors as the future use of the cell ortransgenic animal into which a sequence encoding an engineered RNAprecursor is being introduced, and the level of expression of thedesired RNA precursor. A person skilled in the art would be able tochoose the appropriate regulatory sequence. For example, the transgenicanimals described herein can be used to determine the role of a testpolypeptide or the engineered RNA precursors in a particular cell type,e.g., a hematopoietic cell. In this case, a regulatory sequence thatdrives expression of the transgene ubiquitously, or ahematopoietic-specific regulatory sequence that expresses the transgeneonly in hematopoietic cells, can be used. Expression of the engineeredRNA precursors in a hematopoietic cell means that the cell is nowsusceptible to specific, targeted RNAi of a particular gene. Examples ofvarious regulatory sequences are described below.

The regulatory sequences can be inducible or constitutive. Suitableconstitutive regulatory sequences include the regulatory sequence of ahousekeeping gene such as the α-actin regulatory sequence, or may be ofviral origin such as regulatory sequences derived from mouse mammarytumor virus (MMTV) or cytomegalovirus (CMV).

Alternatively, the regulatory sequence can direct transgene expressionin specific organs or cell types (see, e.g., Lasko et al., 1992, Proc.Natl. Acad. Sci. USA 89:6232). Several tissue-specific regulatorysequences are known in the art including the albumin regulatory sequencefor liver (Pinkert et al., 1987, Genes Dev. 1:268276); the endothelinregulatory sequence for endothelial cells (Lee, 1990, J. Biol. Chem.265:10446-50); the keratin regulatory sequence for epidermis; the myosinlight chain-2 regulatory sequence for heart (Lee et al., 1992, J. Biol.Chem. 267:15875-85), and the insulin regulatory sequence for pancreas(Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515), or thevav regulatory sequence for hematopoietic cells (Oligvy et al., 1999,Proc. Natl. Acad. Sci. USA 96:14943-14948). Another suitable regulatorysequence, which directs constitutive expression of transgenes in cellsof hematopoietic origin, is the murine MHC class I regulatory sequence(Morello et al., 1986, EMBO J. 5:1877-1882). Since NMC expression isinduced by cytokines, expression of a test gene operably linked to thisregulatory sequence can be upregulated in the presence of cytokines.

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals such as mice, include regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG) (collectively referred toas “the regulatory molecule”). Each of these expression systems is welldescribed in the literature and permits expression of the transgenethroughout the animal in a manner controlled by the presence or absenceof the regulatory molecule. For a review of inducible expressionsystems, see, e.g., Mills, 2001, Genes Devel. 15:1461-1467, andreferences cited therein.

The regulatory elements referred to above include, but are not limitedto, the cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus (Bernoist et al., Nature, 290:304, 1981),the tet system, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors. Additional promoters include the promoter contained inthe 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797, 1988); the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. USA 78:1441, 1981); or the regulatory sequencesof the metallothionein gene (Brinster et al., Nature 296:39, 1988).

4. Assay for Testing Engineered RNA Precursors

Drosophila embryo lysates can be used to determine if an engineered RNAprecursor was, in fact, the direct precursor of a mature stRNA or siRNA.This lysate assay is described in Tuschl et al., 1999, supra, Zamore etal., 2000, supra, and Hutvdgner et al. 2001, supra. These lysatesrecapitulate RNAi in vitro, thus permitting investigation into whetherthe proposed precursor RNA was cleaved into a mature stRNA or siRNA byan RNAi-like mechanism. Briefly, the precursor RNA is incubated withDrosophila embryo lysate for various times, and then assayed for theproduction of the mature siRNA or stRNA by primer extension or Northernhybridization. As in the in vivo setting, mature RNA accumulates in thecell-free reaction. Thus, an RNA corresponding to the proposed precursorcan be shown to be converted into a mature stRNA or siRNA duplex in theDrosophila embryo lysate.

Furthermore, an engineered RNA precursor can be functionally tested inthe Drosophila embryo lysates. In this case, the engineered RNAprecursor is incubated in the lysate in the presence of a 5′radiolabeled target mRNA in a standard in vitro RNAi reaction forvarious lengths of time. The target mRNA can be 5′ radiolabeled usingguanylyl transferase (as described in Tuschl et al., 1999, supra andreferences therein) or other suitable methods. The products of the invitro reaction are then isolated and analyzed on a denaturing acrylamideor agarose gel to determine if the target mRNA has been cleaved inresponse to the presence of the engineered RNA precursor in thereaction. The extent and position of such cleavage of the mRNA targetwill indicate if the engineering of the precursor created a pre-siRNAcapable of mediating sequence-specific RNAi.

III. Methods of Introducing RNAs, Vectors, and Host Cells

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell with the target gene may be derived from or contained in anyorganism. The organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. Preferred microbes are those usedin agriculture or by industry, and those that are pathogenic for plantsor animals. Fungi include organisms in both the mold and yeastmorphologies. Plants include arabidopsis; field crops (e.g., alfalfa,barley, bean, corn, cotton, flax, pea, rape, nice, rye, safflower,sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.,almond, apple, apricot, banana, black-berry, blueberry, cacao, cherry,coconut, cranberry, date, faJoa, filbert, grape, grapefruit, guava,kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passionfruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry,strawberry, tangerine, walnut, and watermelon); and ornamentals (e.g.,alder, ash, aspen, azalea, birch, boxwood, camellia, carnation,chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine,redwood, rhododendron, rose, and rubber). Examples of vertebrate animalsinclude fish, mammal, cattle, goat, pig, sheep, rodent, hamster, mouse,rat, primate, and human; invertebrate animals include nematodes, otherworms, drosophila, and other insects.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of RNAi agent may result in inhibitionin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). Quantitation of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

IV. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted target geneexpression or activity. “Treatment”, or “treating” as used herein, isdefined as the application or administration of a therapeutic agent(e.g., a RNA agent or vector or transgene encoding same) to a patient,or application or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, who has a disease or disorder, asymptom of disease or disorder or a predisposition toward a disease ordisorder, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease or disorder, thesymptoms of the disease or disorder, or the predisposition towarddisease.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject atherapeutic agent (e.g., an RNAi agent or vector or transgene encodingsame). Subjects at risk for a disease which is caused or contributed toby aberrant or unwanted target gene expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe target gene aberrancy, such that a disease or disorder is preventedor, alternatively, delayed in its progression. Depending on the type oftarget gene aberrancy, for example, a target gene, target gene agonistor target gene antagonist agent can be used for treating the subject.The appropriate agent can be determined based on screening assaysdescribed herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating targetgene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves contacting a cell capable of expressing targetgene with a therapeutic agent (e.g., an RNAi agent or vector ortransgene encoding same) that is specific for the target gene or protein(e.g., is specific for the mRNA encoded by said gene or specifying theamino acid sequence of said protein) such that expression or one or moreof the activities of target protein is modulated. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target gene polypeptideor nucleic acid molecule. Inhibition of target gene activity isdesirable in situations in which target gene is abnormally unregulatedand/or in which decreased target gene activity is likely to have abeneficial effect.

3. Pharmacogenomics

The therapeutic agents (e.g., an RNAi agent or vector or transgeneencoding same) of the invention can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withaberrant or unwanted target gene activity. In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.). Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach” can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a targetgene polypeptide of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a therapeutic agent of thepresent invention can give an indication whether gene pathways relatedto toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a therapeuticagent, as described herein.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an RNAi agent (or expression vector or transgene encoding same)as described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

V. Pharmaceutical Compositions

The invention pertains to uses of the above-described agents fortherapeutic treatments as described infra. Accordingly, the modulatorsof the present invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, antibody, or modulatorycompound and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfate; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VI. Transgenic Organisms

Engineered RNA precursors of the invention can be expressed intransgenic animals. These animals represent a model system for the studyof disorders that are caused by, or exacerbated by, overexpression orunderexpression (as compared to wildtype or normal) of nucleic acids(and their encoded polypeptides) targeted for destruction by the RNAiagents, e.g., siRNAs and shRNAs, and for the development of therapeuticagents that modulate the expression or activity of nucleic acids orpolypeptides targeted for destruction.

Transgenic animals can be farm animals (pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (for example, baboons, monkeys, andchimpanzees), and domestic animals (for example, dogs and cats).Invertebrates such as Caenorhabditis elegans or Drosophila can be usedas well as non-mammalian vertebrates such as fish (e.g., zebrafish) orbirds (e.g., chickens).

Engineered RNA precursors with stems of 18 to 30 nucleotides in lengthare preferred for use in mammals, such as mice. A transgenic founderanimal can be identified based upon the presence of a transgene thatencodes the new RNA precursors in its genome, and/or expression of thetransgene in tissues or cells of the animals, for example, using PCR orNorthern analysis. Expression is confirmed by a decrease in theexpression (RNA or protein) of the target sequence.

A transgenic founder animal can be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding the RNA precursors can further be bred to othertransgenic animals carrying other transgenes. In addition, cellsobtained from the transgenic founder animal or its offspring can becultured to establish primary, secondary, or immortal cell linescontaining the transgene.

1. Procedures for Making Transgenic, Non-Human Animals

A number of methods have been used to obtain transgenic, non-humananimals, which are animals that have gained an additional gene by theintroduction of a transgene into their cells (e.g., both the somatic andgenn cells), or into an ancestor's genn line. In some cases, transgenicanimals can be generated by commercial facilities (e.g., The TransgenicDrosophila Facility at Michigan State University, The TransgenicZebrafish Core Facility at the Medical College of Georgia (Augusta,Ga.), and Xenogen Biosciences (St. Louis, Mo.). In general, theconstruct containing the transgene is supplied to the facility forgenerating a transgenic animal.

Methods for generating transgenic animals include introducing thetransgene into the germ line of the animal. One method is bymicroinjection of a gene construct into the pronucleus of an early stageembryo (e.g., before the four-cell stage; Wagner et al., 1981, Proc.Natl. Acad. Sci. USA 78:5016; Brinster et al., 1985, Proc. Natl. Acad.Sci. USA 82:4438). Alternatively, the transgene can be introduced intothe pronucleus by retroviral infection. A detailed procedure forproducing such transgenic mice has been described (see e.g., Hogan etal., MP1 ulating the Mouse ErnbnLo. Cold Spring Harbour Laboratory, ColdSpring Harbour, N Y (1986); U.S. Pat. No. 5,175,383 (1992)). Thisprocedure has also been adapted for other animal species (e.g., Hammeret al., 1985, Nature 315:680; Murray et al., 1989, Reprod. Fert. Devl.1:147; Pursel et al., 1987, Vet. hnmunol. Histopath. 17:303; Rexroad etal., 1990, J. Reprod. Fert. 41 (suppl): 1 19; Rexroad et al., 1989,Molec. Reprod. Devl. 1:164; Simons et al., 1988, BioTechnology 6:179;Vize et al., 1988, J. Cell. Sci. 90:295; and Wagner, 1989, J. Cell.Biochem. 13B (suppl): 164).

In brief, the procedure involves introducing the transgene into ananimal by microinjecting the construct into the pronuclei of thefertilized mammalian egg(s) to cause one or more copies of the transgeneto be retained in the cells of the developing mammal(s). Followingintroduction of the transgene construct into the fertilized egg, the eggmay be incubated in vitro for varying amounts of time, or reimplanted ain surrogate host, or both. One common method is to incubate the embryosin vitro for about 1-7 days, depending on the species, and thenreimplant them into the surrogate host. The presence of the transgene inthe progeny of the transgenically manipulated embryos can be tested bySouthern blot analysis of a segment of tissue.

Another method for producing germ-line transgenic animals is through theuse of embryonic stem (ES) cells. The gene construct can be introducedinto embryonic stem cells by homologous recombination (Thomas et al.,1987, Cell 51:503; Capecchi, Science 1989, 244:1288; Joyner et al.,1989, Nature 338:153) in a transcriptionally active region of thegenome. A suitable construct can also be introduced into embryonic stemcells by DNA-mediated transfection, such as by 17 electroporation(Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, 1987). Detailed procedures for culturing embryonic stem cells(e.g., ES-D3@ ATCC # CCL-1934, ES-El4TG2a, ATCC # CCL-1821, AmericanType Culture Collection, Rockville, AM) and methods of making transgenicanimals from embryonic stem cells can be found in Teratocarcinomas andEmbi3Lonic Stem Cells, A Practical Approach, ed. E. J. Robertson (IRLPress, 1987). In brief, the ES cells are obtained from pre-implantationembryos cultured in vitro (Evans et al., 1981, Nature 292:154-156).Transgenes can be efficiently introduced into ES cells by DNAtransfection or by retrovirus-mediated transduction. The resultingtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells colonize the embryo and contribute to thegerm line of the resulting chimeric animal.

In the above methods, the transgene can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can beincorporated and inherited as a transgene integrated into the hostgenome. The transgene can also be constructed to permit it to beinherited as an extrachromosomal plasmid (Gassmann et al., 1995, Proc.Natl. Acad. Sci. USA 92:1292). A plasmid is a DNA molecule that canreplicate autonomously in a host.

The transgenic, non-human animals can also be obtained by infecting ortransfecting cells either in vivo (e.g., direct injection), ex vivo(e.g., infecting the cells outside the host and later reimplanting), orin vitro (e.g., infecting the cells outside host), for example, with arecombinant viral vector carrying a gene encoding the engineered RNAprecursors. Examples of suitable viral vectors include recombinantretroviral vectors (Valerio et al., 1989, Gene 84:419; Scharfinan etal., 1991, Proc. Natl. Acad. Sci. USA 88:462; Miller and Buttimore,1986, Mol. Cell. Biol. 6:2895), recombinant adenoviral vectors (Freidmanet al., 1986, Mol. Cell. Biol. 6:3791; Levrero et al., 1991, Gene 101:195), and recombinant Herpes simplex viral vectors (Fink et al., 1992,Human Gene Therapy 3:11). Such methods are also useful for introducingconstructs into cells for uses other than generation of transgenicanimals.

Other approaches include insertion of transgenes encoding the newengineered RNA precursors into viral vectors including recombinantadenovirus, adenoassociated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly. Other approaches include delivering the transgenes, inthe form of plasmid DNA, with the help of, for example, cationicliposomes (lipofectin) or derivatized (e.g., antibody conjugated)polylysine conjugates, gramacidin S, artificial viral envelopes, orother such intracellular carriers, as well as direct injection of thetransgene construct or CaPO₄ precipitation carried out in vivo. Suchmethods can also be used in vitro to introduce constructs into cells foruses other than generation of transgenic animals.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gone delivery system for the transfer of exogenous genes invivo or in vitro. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host. The development of specialized cell lines(termed “packaging cells”) which produce only replication-defectiveretroviruses has increased the utility of retroviruses for gene therapy,and defective retroviruses are characterized for use in gene transferfor gene therapy purposes (for a review see Miller, 1990, Blood 76:271).A replication defective retrovirus can be packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.,(eds.) Greene Publishing Associates, (1989), Sections 9 9.14 and otherstandard laboratory manuals.

Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare known to those skilled in the art. Examples of suitable packagingvirus lines for preparing both ecotropic and amphotropic retroviralsystems include Psi-Crip, PsiCre, Psi-2 and Psi-Am. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al., 1985, Science 230:1395-1398; Danos andMulligan, 1988, Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.,1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990,Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl.Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci.USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; andPCT Application WO 92/07573).

In another example, recombinant retroviral vectors capable oftransducing and expressing genes inserted into the genome of a cell canbe produced by transfecting the recombinant retroviral genome intosuitable packaging cell lines such as PA317 and Psi-CRIP (Comette etal., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl.Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used toinfect a wide variety of cells and tissues in susceptible hosts (e.g.,rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. InfectiousDisease, 166:769), and also have the advantage of not requiringmitotically active cells for infection. Another viral gene deliverysystem useful in the present invention also utilizes adenovirus-derivedvectors. The genome of an adenovirus can be manipulated such that itencodes and expresses a gene product of interest but is inactivated interms of its ability to replicate in a normal lytic viral life cycle.See, for example, Berkner et al. (1988, BioTechniques 6:616), Rosenfeldet al. (1991, Science 252:431-434), and Rosenfeld et al. (1992, Cell68:143-155). Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, AO,Ad7 etc.) are known to those skilled in the art. Recombinantadenoviruses can be advantageous in certain circumstances in that theyare not capable of infecting nondividing cells and can be used to infecta wide variety of cell types, including epithelial cells (Rosenfeld etal., 1992, cited supra). Furthermore, the virus particle is relativelystable and amenable to purification and concentration, and as above, canbe modified to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis hz situ where introduced DNA becomes integrated into thehost genome (e.g., retroviral DNA). Moreover, the carrying capacity ofthe adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham, 1986, J. Virol. 57:267).

Yet another viral vector system useful for delivery of the subjecttransgenes is the adeno-associated virus (AAV). Adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. For a review, see Muzyczka etal. (1992, Curr. Topics in Micro. and Immunol. 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992, Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al., 1989, J. Virol. 63:3822-3828; and McLaughlin et al.(1989, J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) MoL Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHennonat et al. (1984) Proc. Nad. Acad. Sci. USA 8 1:64666470; Tratschinet al. (1985) Mol. Cell. BioL 4:2072-2081; Wondisford et al. (1988) MoLEndocrinoL 2:32-39; Tratschin et al. (1984) J ViroL 51:611-619; andFlotte et al. (1993) J BioL Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of an shRNAor engineered RNA precursor of the invention in the tissue of an animal.Most non-viral methods of gene transfer rely on nominal mechanisms usedby mammalian cells for the uptake and intracellular transport ofmacromolecules. In preferred embodiments, non-viral gene deliverysystems of the present invention rely on endocytic pathways for theuptake of the subject gene of the invention by the targeted cell.Exemplary gene delivery systems of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes. Otherembodiments include plasmid injection systems such as are described inMeuli et al., (2001) J Invest. DerinatoL, 116(1):131-135; Cohen et al.,(2000) Gene Ther., 7(22):1896-905; and Tam et al., (2000) Gene Ther.,7(21):186774.

In a representative embodiment, a gene encoding an shRNA or engineeredRNA precursor of the invention can be entrapped in liposomes bearingpositive charges on their surface (e.g., lipofectins) and (optionally)which are tagged with antibodies against cell surface antigens of thetarget tissue (Mizuno et al., (1992) No Shinkei Geka, 20:547-55 1; PCTpublication WO91/06309; Japanese patent application 10473 8 1; andEuropean patent publication EP-A-43 075).

Animals harboring the transgene can be identified by detecting thepresence of the transgene in genomic DNA (e.g., using Southernanalysis). In addition, expression of the shRNA or engineered RNAprecursor can be detected directly (e.g., by Northern analysis).Expression of the transgene can also be confirmed by detecting adecrease in the amount of protein corresponding to the targetedsequence. When the transgene is under the control of an inducible ordevelopmentally regulated promoter, expression of the target protein isdecreased when the transgene is induced or at the developmental stagewhen the transgene is expressed, respectively.

2. Clones of Transgenic Animals

Clones of the non-human transgenic animals described herein can beproduced according to the methods described in Wilmut et al. ((1997)Nature, 385:810-813) and PCT publication Nos. WO 97/07668 and WO97/07669. In brief, a cell, e.g., a somatic cell from the transgenicanimal, can be isolated and induced to exit the growth cycle and enterthe GO phase to become quiescent. The quiescent cell can then be fused,e.g., through the use of electrical pulses, to an enucleated oocyte froman animal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops into amorula or blastocyte and is then transferred to a pseudopregnant femalefoster animal. Offspring borne of this female foster animal will beclones of the animal from which the cell, e.g., the somatic cell, wasisolated.

Once the transgenic animal is produced, cells of the transgenic animaland cells from a control animal are screened to determine the presenceof an RNA precursor nucleic acid sequence, e.g., using polymerase chainreaction (PCR). Alternatively, the cells can be screened to determine ifthe RNA precursor is expressed (e.g., by standard procedures such asNorthern blot analysis or reverse transcriptase-polymerase chainreaction (RT-PCR); Sambrook et al., Molecular Cloning—A LaboratoryManual, (Cold Spring Harbor Laboratory, 1989)).

The transgenic animals of the present invention can be homozygous orheterozygous, and one of the benefits of the invention is that thetarget mRNA is effectively degraded even in heterozygotes. The presentinvention provides for transgenic animals that carry a transgene of theinvention in all their cells, as well as animals that carry a transgenein some, but not all of their cells. That is, the invention provides formosaic animals. The transgene can be integrated as a single transgene orin concatatners, e.g., head-to-head tandems or head-to-tail tandems.

For a review of techniques that can be used to generate and assesstransgenic animals, skilled artisans can consult Gordon (IwL Rev. CytoL1 1 5:171-229, 1989), and may obtain additional guidance from, forexample: Hogan et al. “Manipulating the Mouse Embryo” (Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1986; Krimpenfort et al.,BiolTechnology 9:86, 1991; Palmiter et al., Cell 41:343, 1985; Kraemeret al., “Genetic Manipulation of the Early Mammalian Embryo,” ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1985; Hammer et al.,Nature 315:680, 1985; Purcel et al., Science, 244:1281, 1986; Wagner etal., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S. Pat. No.5,175,384.

3. Transgenic Plants

Among the eukaryotic organisms featured in the invention are plantscontaining an exogenous nucleic acid that encodes an engineered RNAprecursor of the invention.

Accordingly, a method according to the invention comprises making aplant having a nucleic acid molecule or construct, e.g., a transgene,described herein. Techniques for introducing exogenous micleic acidsinto monocotyledonous and dicotyledonous plants are known in the art,and include, without limitation, Agrobacterium-mediated transformation,viral vector-mediated transformation, electroporation and particle guntransformation, see, e.g., U.S. Pat. Nos. 5,204,253 and 6,013,863. If acell or tissue culture is used as the recipient tissue fortransformation, plants can be regenerated from transformed cultures bytechniques known to those skilled in the art. Transgenic plants can beentered into a breeding program, e.g., to introduce a nucleic acidencoding a polypeptide into other lines, to transfer the nucleic acid toother species or for further selection of other desirable traits.Alternatively, transgenic plants can be propagated vegetatively forthose species amenable to such techniques. Progeny includes descendantsof a particular plant or plant line. Progeny of a plant include seedsformed on F1, F2, F3, and subsequent generation plants, or seeds formedon BQ, BC2, BC3, and subsequent generation plants. Seeds produced by atransgenic plant can be grown and then selfed (or outcrossed and selfed)to obtain seeds homozygous for the nucleic acid encoding a novelpolypeptide.

A suitable group of plants with which to practice the invention includedicots, such as safflower, alfalfa, soybean, rapeseed (high erucic acidand canola), or sunflower. Also suitable are monocots such as corn,wheat, rye, barley, oat, rice, millet, amaranth or sorghum. Alsosuitable are vegetable crops or root crops such as potato, broccoli,peas, sweet corn, popcorn, tomato, beans (including kidney beans, limabeans, dry beans, green beans) and the like. Also suitable are fruitcrops such as peach, pear, apple, cherry, orange, lemon, grapefruit,plum, mango and palm. Thus, the invention has use over a broad range ofplants, including species from the genera Anacardium, Arachis,Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum,Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria,Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyalnus,Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana,Medicago, Nicotiana, Olea, Oryza, Panicum, Pannesetum, Persea,Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum,Vicia, Vitis, Vigna and Zea.

The skilled artisan will appreciate that the enumerated organisms arealso useful for practicing other aspects of the invention, e.g., as hostcells, as described supra.

The nucleic acid molecules of the invention can be expressed in plantsin a cell- or tissue-specific manner according to the regulatoryelements chosen to include in a particular nucleic acid constructpresent in the plant. Suitable cells, tissues, and organs in which toexpress a chimeric polypeptide of the invention include, withoutlimitation, egg cell, central cell, synergid cell, zygote, ovuleprimordia, nucellus, integuments, endothelium, female gametophyte cells,embryo, axis, cotyledons, suspensor, endosperm, seed coat, groundmeristem, vascular bundle, cambium, phloem, cortex, shoot or root apicalmeristems, lateral shoot or root meristems, floral meristem, leafprimordia, leaf mesophyll cells, and leaf epidermal cells, e.g.,epidermal cells involved in fortning the cuticular layer. Also suitableare cells and tissues grown in liquid media or on semi-solid media.

4. Transgenic Fungi

Other eukaryotic organisms featured in the invention are fungicontaining an exogenous nucleic acid molecule that encodes an engineeredRNA precursor of the invention. Accordingly, a method according to theinvention comprises introducing a nucleic acid molecule or construct asdescribed herein into a fungus. Techniques for introducing exogenousnucleic acids into many fungi are known in the art, see, e.g., U.S. Pat.Nos. 5,252,726 and 5,070,020. Transformed fungi can be cultured bytechniques known to those skilled in the art. Such fungi can be used tointroduce a nucleic acid encoding a polypeptide into other fungalstrains, to transfer the nucleic acid to other species or for ftirtherselection of other desirable traits.

A suitable group of fungi with which to practice the invention includefission yeast and budding yeast, such as Saccharomyces cereviseae, S.pombe, S. carlsbergeris and Candida albicans. Filamentous fungi such asAspergillus spp. and Penicillium spp. are also useful.

VII. Knockout and/or Knockdown Cells or Organisms

A further preferred use for the RNAi agents of the present invention (orvectors or transgenes encoding same) is a functional analysis to becarried out in eukaryotic cells, or eukaryotic non-human organisms,preferably mammalian cells or organisms and most preferably human cells,e.g., cell lines such as HeLa or 293 or rodents, e.g., rats and mice. Byadministering a suitable RNAi agent which is sufficiently complementaryto a target mRNA sequence to direct target-specific RNA interference, aspecific knockout or knockdown phenotype can be obtained in a targetcell, e.g., in cell culture or in a target organism.

Thus, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout or knockdown phenotype comprising a fully or at least partiallydeficient expression of at least one endogeneous target gene whereinsaid cell or organism is transfected with at least one vector comprisingDNA encoding a RNAi agent capable of inhibiting the expression of thetarget gene. It should be noted that the present invention allows atarget-specific knockout or knockdown of several different endogeneousgenes due to the specificity of the RNAi agent.

Gene-specific knockout or knockdown phenotypes of cells or non-humanorganisms, particularly of human cells or non-human mammals may be usedin analytic to procedures, e.g., in the functional and/or phenotypicalanalysis of complex physiological processes such as analysis of geneexpression profiles and/or proteomes. Preferably the analysis is carriedout by high throughput methods using oligonucleotide based chips.

Using RNAi based knockout or knockdown technologies, the expression ofan endogeneous target gene may be inhibited in a target cell or a targetorganism. The endogeneous gene may be complemented by an exogenoustarget nucleic acid coding for the target protein or a variant ormutated form of the target protein, e.g., a gene or a DNA, which mayoptionally be fused to a further nucleic acid sequence encoding adetectable peptide or polypeptide, e.g., an affinity tag, particularly amultiple affinity tag.

Variants or mutated forms of the target gene differ from the endogeneoustarget gene in that they encode a gene product which differs from theendogeneous gene product on the amino acid level by substitutions,insertions and/or deletions of single or multiple amino acids. Thevariants or mutated forms may have the same biological activity as theendogeneous target gene. On the other hand, the variant or mutatedtarget gene may also have a biological activity, which differs from thebiological activity of the endogeneous target gene, e.g., a partiallydeleted activity, a completely deleted activity, an enhanced activityetc. The complementation may be accomplished by compressing thepolypeptide encoded by the endogeneous nucleic acid, e.g., a fusionprotein comprising the target protein and the affinity tag and thedouble stranded RNA molecule for knocking out the endogeneous gene inthe target cell. This compression may be accomplished by using asuitable expression vector expressing both the polypeptide encoded bythe endogenous nucleic acid, e.g., the tag-modified target protein andthe double stranded RNA molecule or alternatively by using a combinationof expression vectors. Proteins and protein complexes which aresynthesized de novo in the target cell will contain the exogenous geneproduct, e.g., the modified fusion protein. In order to avoidsuppression of the exogenous gene product by the RNAi agent, thenucleotide sequence encoding the exogenous nucleic acid may be alteredat the DNA level (with or without causing mutations on the amino acidlevel) in the part of the sequence which so is homologous to the RNAiagent. Alternatively, the endogeneous target gene may be complemented bycorresponding nucleotide sequences from other species, e.g., from mouse.

VIII. Functional Genomics and/or Proteomics

Preferred applications for the cell or organism of the invention is theanalysis of gene expression profiles and/or proteomes. In an especiallypreferred embodiment an analysis of a variant or mutant form of one orseveral target proteins is carried out, wherein said variant or mutantforms are reintroduced into the cell or organism by an exogenous targetnucleic acid as described above. The combination of knockout of anendogeneous gene and rescue by using mutated, e.g., partially deletedexogenous target has advantages compared to the use of a knockout cell.Further, this method is particularly suitable for identifying functionaldomains of the targeted protein. In a further preferred embodiment acomparison, e.g., of gene expression profiles and/or proteomes and/orphenotypic characteristics of at least two cells or organisms is carriedout. These organisms are selected from: (i) a control cell or controlorganism without target gene inhibition, (ii) a cell or organism withtarget gene inhibition and (iii) a cell or organism with target geneinhibition plus target gene complementation by an exogenous targetnucleic acid.

Furthermore, the RNA knockout complementation method may be used for ispreparative purposes, e.g., for the affinity purification of proteins orprotein complexes from eukaryotic cells, particularly mammalian cellsand more particularly human cells. In this embodiment of the invention,the exogenous target nucleic acid preferably codes for a target proteinwhich is fused to art affinity tag. This method is suitable forfunctional proteome analysis in mammalian cells, particularly humancells.

Another utility of the present invention could be a method ofidentifying gene function in an organism comprising the use of an RNAiagent to inhibit the activity of a target gene of previously unknownfunction. Instead of the time consuming and laborious isolation ofmutants by traditional genetic screening, functional genomics wouldenvision determining the function of uncharacterized genes by employingthe invention to reduce the amount and/or alter the timing of targetgene activity. The invention could be used in determining potentialtargets for pharmaceutics, understanding normal and pathological eventsassociated with development, determining signaling pathways responsiblefor postnatal development/aging, and the like. The increasing speed ofacquiring nucleotide sequence information from genomic and expressedgene sources, including total sequences for the yeast, D. melanogaster,and C. elegans genomes, can be coupled with the invention to determinegene function in an organism (e.g., nematode). The preference ofdifferent organisms to use particular codons, searching sequencedatabases for related gene products, correlating the linkage map ofgenetic traits with the physical map from which the nucleotide sequencesare derived, and artificial intelligence methods may be used to defineputative open reading frames from the nucleotide sequences acquired insuch sequencing projects. A simple assay would be to inhibit geneexpression according to the partial sequence available from an expressedsequence tag (EST). Functional alterations in growth, development,metabolism, disease resistance, or other biological processes would beindicative of the normal role of the EST's gene product.

The ease with which RNA can be introduced into an intact cell/organismcontaining the target gene allows the present invention to be used inhigh throughput screening (HTS). Solutions containing RNAi agents thatare capable of inhibiting the different expressed genes can be placedinto individual wells positioned on a microtiter plate as an orderedarray, and intact cells/organisms in each well can be assayed for anychanges or modifications in behavior or development due to inhibition oftarget gene activity. The amplified RNA can be fed directly to, injectedinto, the cell/organism containing the target gene. Alternatively, theRNAi agent can be produced from a vector, as described herein. Vectorscan be injected into, the cell/organism containing the target gene. Thefunction of the target gene can be assayed from the effects it has onthe cell/organism when gene activity is inhibited. This screening couldbe amenable to small subjects that can be processed in large number, forexample: arabidopsis, bacteria, drosophila, fungi, nematodes, viruses,zebrafish, and tissue culture cells derived from mammals. A nematode orother organism that produces a colorimetric, fluorogenic, or luminescentsignal in response to a regulated promoter (e.g., transfected with areporter gene construct) can be assayed in an HTS format.

The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of RNAi agentsat specific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

IX. Screening Assays

The methods of the invention are also suitable for use in methods toidentify and/or characterize potential pharmacological agents, e.g.,identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents.

Thus, the present invention also relates to a system for identifyingand/or characterizing pharmacological agents acting on at least onetarget protein comprising: (a) a eukaryotic cell or a eukaryoticnon-human organism capable of expressing at least one endogeneous targetgene coding for said so target protein, (b) at least one RNA agentcapable of inhibiting the expression of said at least one endogeneoustarget gene, and (c) a test substance or a collection of test substanceswherein pharmacological properties of said test substance or saidcollection are to be identified and/or characterized. Further, thesystem as described above preferably comprises: (d) at least oneexogenous target nucleic acid coding for the target protein or a variantor mutated form of the target protein wherein said exogenous targetnucleic acid differs from the endogeneous target gene on the nucleicacid level such that the expression of the exogenous target nucleic acidis substantially less inhibited by the RNA agent than the expression ofthe endogeneous target gene.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner USP 409), plasmids (Cull et al.(1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith(1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra.)).

In a preferred embodiment, the library is a natural product library,e.g., a library produced by a bacterial, fungal, or yeast culture. Inanother preferred embodiment, the library is a synthetic compoundlibrary.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Materials and Methods Lysate Preparation

Fly Embryo Lysates were Prepared as Previously Described (Tuschl et al.1999). Wheat germ extracts were prepared from frozen or vacuum-packedraw wheat germ (e.g., Fearn Nature Fresh Raw Wheat Germ, Bread andCircus) as described (Erickson and Blobel 1983). The extract wascentrifuged at 14,500 g at 4° C. for 25 min; the supernatant was thenfrozen in aliquots in liquid nitrogen and stored at −80° C. Forcauliflower extract, the outer layer of fresh cauliflower (ShawsSupermarket) was harvested with a razor blade and ground to a powderunder liquid nitrogen in a mortar and pestle, then homogenized with 3 mLof 1×lysis buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate) containing 5 mM dithiothreitol (DTT) and 1 mg/mLPefabloc SC (Boehringer Mannheim) per gram of plant tissue. The extractwas centrifuged, and the supernatant was stored as described for theDrosophila embryo lysate.

Analysis of dsRNA Processing

For analysis of dsRNA processing, 5 nM internally α-32P-UTP-labeleddsRNA was incubated in a 10-4 reaction containing 5 μL of Drosophilaembryo lysate (Tuschl et al. 1999) or wheat germ extract, 100 μM GTP,500 μM ATP, 10 mM creatine phosphate, 10 μg/mL creatine phosphokinase, 5mM DTT, and 0.1 U/μL RNasin (Promega) at 25° C. for 3 h. Reactions werestopped by the addition of 2× proteinase K buffer [200 mM Tris-HCl at pH7.5, 25 mM EDTA, 300 mM NaCl, 2% (w/v) sodium dodecyl sulfate] anddeproteinized with mg/mL proteinase K at 65° C. for 15 min. Productswere precipitated with 3 volumes cold ethanol and analyzed byelectrophoresis in a 15% polyacrylamide sequencing gel.

Gel Filtration and RNAse Protection

Internally α-32P-UTP-labeled dsRNAs were incubated in wheat germextract, then deproteinized at room temperature with proteinase K (1 h)and RNA-precipitated with 3 volumes of cold ethanol. The RNA wasresuspended in 1× lysis buffer and analyzed by gel filtration asdescribed (Nykanen et al. 2001). For RNase protection, the RNA productsof a 10-μL wheat germ extract reaction were deproteinized at roomtemperature and analyzed by RNAse protection essentially as described(Sambrook et al. 1989). Briefly, the siRNA pellets were dissolved in 10μL of RNAse digestion buffer (300 mM NaCl, 10 mM Tris-HCl at pH 7.4, and5 mM EDTA at pH 7.5) containing 10 mM [beta]-glycerophosphate, 5 mM ATP,0-6.6 U of RNAse A, and 0-1.1 U of RNAse T1. For control experiments,5′-32P-radiolabeled synthetic, double-stranded siRNAs were mixed withthe products of a wheat germ reaction performed with unlabeled dsRNA andcoprecipitated with 3 volumes of cold ethanol. RNAse protection was at25° C. for 1 h, stopped by adding 0.6 μL of 10% SDS and 0.3 μL of 20mg/mL proteinase K, then incubated at 25° C. for 1 h. The reactions werethen adjusted to 200 μL with 2×PK buffer containing 0.2 mg/mL Glycogen(Roche), extracted with an equal volume ofphenol/chloroform/isoamylalcohol (25:24:1; v/v/v), precipitated with 3volumes of cold ethanol, and analyzed in a 15% sequencing polyacrylamidegel.

Synthetic siRNAs Used as Inhibitors

The 21-nt siRNA inhibitor comprised CGUACGCGGAAUAC UUCGA(5-Iodo-U)Uannealed with UCGAAGUAUUCCGCGUACGUG; the 25-mer comprisedAUCACGUACGCGGAAUACUUCGA(5-Iodo-U)U annealed withUCGAAGUAUUCCGCGUACGUGAUUG. The 5-Iodo-U nucleotides were included tofacilitate studies not presented here, and we have no evidence theyenhance the effectiveness of the siRNAs as inhibitors.

Analysis of RdRP Activity

Assays were performed in a final volume of 10 μL containing 5 μL oflysate, 100 μM GTP, 100 μM CTP, 500 μM ATP, 20 μM UTP, 5 μCi ofα-32P-UTP (25 Ci/mmole), 10 mM creatine phosphate, 10 μg/mL creatinephosphokinase, 5 mM DTT, 0.2 U/4 Super-RNasin (Ambion), and 7-methyl-G-or A-capped RNAs. After incubation at 25° C. for 3 h, the reaction wasdeproteinized with proteinase K in 200 μL of 2× proteinase K buffer at65° C. for 15 min. After phenol/chloroform/isoamylalcohol extraction,the aqueous phase was precipitated with 3 volumes of cold ethanol,resuspended in 10 μL of 2× formamide loading buffer as described(Sambrook et al. 1989), and resolved on 10% or 15% polyacrylamidesequencing gels. For primed assays, capped RNAs were preincubated withsingle-stranded 21-nt RNA primers or siRNA duplexes at room temperaturefor 10 min before the remaining reaction components were added.

Arabidopsis PHV, PHB, and Mutant PHV Target RNAs

Arabidopsis PHV and PHB cDNA sequences containing the miR165/166complementary sequences were amplified from an Arabidopsis flower cDNAlibrary (CD4-6) by polymerase chain reaction (PCR) using the followingprimer pairs: 5′-PHV primer, GCGTAATACGACTCACTATAGGCGCCGGAACAAGTTG AAG(SEQ ID NO:7), and 3′-PHV primer, GACAGTCACGGAACCAAGATG (SEQ ID NO:8);or 5′-PHB primer, GCGTAATACGACTCACTATAGGTGAGTCTGTGGTCGTGAGTG (SEQ IDNO:9), and 3′-PHB primer, GCTGCTGCTAAAGTCGTAGGA (SEQ ID NO:10). TheArabidopsis G [right-arrow]. A mutant phv template was initiallyamplified using the 5′-PHV primer andCCACTGCAGTTGCGTGAAACAGCTACGATACCAATAGAATCCGGATCAGGC TTCATCCC (SEQ IDNO:11). This PCR product was diluted 100-fold, then reamplified with the5′-PHV primer and GACAGTCACGGAACCAAGATGGACGATCTTTGAGGATTTCAGCGACCTTCATGGGTTCTAAACTCACGAGGCCACAGGCACGTGCTGCTATTCCACTGCAGTTG CGTGAAACAGC (SEQ IDNO:12). In vitro RNA transcription and cap labeling were as described(Tuschl et al. 1999; Zamore et al. 2000).

In Vitro RNAi in Fly Embryo Lysate and Wheat Germ Extract

For RNAi in Drosophila embryo lysate, four siRNA duplexes werechemically synthesized (Dharmacon), annealed, and incubated in astandard RNAi reaction (Zamore et al. 2000). The sequences of siRNAs(sense and antisense strands) corresponding to miR165, miR166, PHV, andmutant phv target positions were miR165, UCGGACCAGGCUUCAUCCCCC (SEQ IDNO:13) and GGGAUGAAGCCUGGUCCGAGG (SEQ ID NO:14); miR166,UCGGACCAGGCUUCAUUCCCC (SEQ ID NO:15) and GGAAUGAAGCCUGGUCCGAGA (SEQ IDNO:16); PHV, CCGGACCAGGCUUCAUCCCAA (SEQ ID NO:17) andGGGAUGAAGCCUGGUCCGGAU (SEQ ID NO:18); and mutant phv,CCGGAUCAGGCUUCAUCCCAA (SEQ ID NO:19) and GGGAUGAAGCCUGAUCCGGAU (SEQ IDNO:20). Wheat germ extract target cleavage reactions were as standardDrosophila in vitro RNAi reactions, except that no exogenous siRNAs wereadded.

Total RNA Isolation and Northern Analysis

Total RNA was isolated from lysates, and Northern analysis was performedas described (Hutvagner and Zamore 2002). 5′-32P-radioalabeled syntheticmiR165 antisense siRNA (above) was used as probe.

Overview of Examples I-VI

The data presented in Examples I-VI demonstrate that extracts of wheatgerm, introduced for the study of translation and protein translocationin the 1970s (Roberts and Paterson 1973), recapitulate many of the keyfeatures of RNA silencing in plants. Using this in vitro system, it isshown that in plants, ATP-dependent, Dicer-like enzymes cleave dsRNAinto small RNAs that have the structure of siRNAs. Unlike Drosophilaembryos or mammalian cells, plants convert dsRNA into two distinctclasses of siRNAs, long and short siRNAs. Inhibitor studies indicatethat a different Dicer-like enzyme generates each siRNA class.

The data further demonstrate that a wheat RdRP activity can synthesizedsRNA using exogenous single-stranded RNA as a template without anexogenous primer, and that this dsRNA is preferentially converted intolong siRNAs.

Finally, it is demonstrated that wheat germ extracts contain anendogenous RISC programmed with a miRNA. This endogenous miRNA complexcan direct efficient cleavage of the wild-type Arabidopsis PHAVOLUTA(PHV) mRNA sequence, but not that of a previously described dominant PHVmutant that perturbs leaf development. This finding supports the viewthat in plants miRNAs direct RNAi and explains the molecular basis forthe dominant PHV mutation in Arabidopsis. Interestingly, exactcomplementarity between the miRNA and target mRNA is not necessary forthe miRNA to direct efficient target cleavage. In fact, it isdemonstrated that the efficiency of cleavage is greater when a G:U basepair, referred to also as a G:U wobble, is present near the 5′ or 3′ endof the complex formed between the miRNA and the target. Understandingthe natural mechanism by which miRNAs efficiency mediate RNAi in plantsallows for the design of improved RNAi agents for use in mediating RNAinot only in plants, but in eukaryotes (in particular, in mammals).

Example I: Two Distinct Classes of Small RNAs Derived from dsRNA inPlant Extracts

Two distinct classes of small RNAs are produced in transgenic plantsbearing silenced transgenes (Hamilton et al. 2002; Mallory et al. 2002).To test if the production of these two classes of small RNAs was anormal feature of plant biology or a specialized response to foreignDNA, the length distribution of a nonredundant set of 423 endogenoussmall RNAs cloned from Arabidopsis thaliana was examined. (For thesequences of 143 of the small RNAs see Llave et al. 2002a; Reinhart etal. 2002). Excluded from this analysis are cloned fragments of tRNA andrRNA. Included in the set are known and predicted miRNAs, as well assmall RNAs of unknown function corresponding to intragenic regions or tomRNA sequences in either the sense or antisense orientation. Thedistribution of lengths within this set was bimodal, with peaks at 21and 24 nt (FIG. 1A). In contrast, the length distribution of clonedsmall RNAs from C. elegans forms a single broad peak (Lau et al. 2001).The two classes of green fluorescent protein (GFP)-derived small RNAswere proposed to be siRNAs with distinct RNA silencing functions: the˜21-mers to direct posttranscriptional silencing via mRNA degradationand the ˜24-mers to trigger systemic silencing and the methylation ofhomologous DNA (Hamilton et al. 2002). Analysis of the two classes ofendogenous small RNAs indicates that each class has a distinct sequencebias, with a 5′-uridine predominating in the shorter class and a5′-adenosine in the longer class (FIG. 1B). The 5′ sequence bias of theshort class is produced by the inclusion in the data set of miRNAs,which in plants and animals typically begin with uridine (Lagos-Quintanaet al. 2001, 2002; Lau et al. 2001; Lee and Ambros 2001; Reinhart et al.2002). Thus, the non-miRNA small RNAs in the shorter class display no 5′sequence bias, whereas a 5′-adenosine is overrepresented in the longerclass. The two classes are either generated by different enzymes,function in separate effector complexes, or both.

Example II: Plant Small RNAs are Bona Fide siRNAs

Although the small RNAs that correlate with the posttranscriptionalsilencing of homologous target mRNAs were first discovered in plants(Hamilton and Baulcombe 1999), they have not yet been shown to be thedirect products of endonucleolytic cleavage of long dsRNA. To begin totest if small RNAs are, in fact, siRNAs, we prepared plant extracts andmonitored them for Dicer-like activity. When uniformly 32P-radiolabeleddsRNA was incubated in wheat germ extract, it was efficiently cleavedinto small RNAs (FIG. 2A). As reported previously for extracts ofDrosophila (Zamore et al. 2000) and for purified Drosophila (Bernsteinet al. 2001) and human Dicer (Billy et al. 2001), no intermediateproducts were detected in the conversion of dsRNA into small RNAs.Unlike the fly and human Dicer reactions, two discrete size classes, one˜21-nt and the other 24-25-nt long, were produced from the dsRNA uponincubation in wheat germ extract (FIG. 2B). The ratio of wheat24-25-mers to −21-mers in 14 separate reactions was 4±1.7, similar tothe roughly 2.5-fold excess of longer small RNA sequences cloned fromArabidopsis. (The 2.5-fold excess of long to short, cloned endogenoussmall RNAs underestimates the ratio, because it includes miRNAs, whichare predominantly short.). Silencing-related small RNAs have thus faronly been demonstrated in vivo for dicots, and wheat is a monocot.Extracts of the dicot cauliflower, a member of the mustard family likeArabidopsis, also converted dsRNA into two discrete sizes of small RNAs,˜21 and ˜24 nt (FIG. 2C). In both Drosophila and C. elegans, Dicerrequires ATP for efficient production of both siRNAs (Zamore et al.2000; Bernstein et al. 2001; Nykanen et al. 2001) and miRNAs (Hutvagneret al. 2001; Ketting et al. 2001). Consistent with the idea that bothclasses of small RNAs are produced by plant orthologs of Dicer,efficient production of both the ˜21-nt and the ˜24-nt small RNAs inwheat germ extract required ATP (FIG. 2D).

Although small, silencing-associated RNAs in plants are commonly calledsiRNAs, and synthetic siRNA duplexes initiate plant RNA silencing(Klahre et al. 2002), plant small RNAs have not been demonstrated to bedouble-stranded RNAs with 2-nt, 3′ overhanging ends and 3′-hydroxyltermini. Such attributes reflect the unique production of siRNAs bymembers of the Dicer family of ribonuclease III enzymes. To determine ifthe small RNAs generated from dsRNA in wheat germ extracts were bonafide siRNAs, we analyzed their structure. Uniformly 32P-radiolabeleddsRNA was incubated in wheat germ extract, deproteinized, andfractionated by gel filtration to resolve single-stranded fromdouble-stranded siRNA (Nykanen et al. 2001). Both classes of small RNAproducts of the in vitro wheat germ reaction comigrated with a syntheticsiRNA duplex and with Drosophila siRNA duplexes generated by processingdsRNA in Drosophila embryo lysate (FIG. 2E). Therefore, the small RNAsgenerated by incubating dsRNA in wheat germ extract are double-stranded.

Next, we examined the end structure of the small RNAs. Treatment of5′-32P-radiolabeled, synthetic siRNA duplexes with the single-strandedRNA-specific nucleases T1 and RNase A removes the 2-nt, 3′ overhangingends typical of siRNAs, generating 1-nt and 2-nt shorter RNAs. In adenaturing polyacrylamide gel, such nuclease products of siRNAs migratefaster, because they contain 3′-phosphates (diagramed in FIG. 2F). Whensynthetic 25-nt duplexes with 2-nt, 3′ overhangs were digested with T1and RNase A, the expected 24-nt and 23-nt, 3′ phosphorylated productswere generated (FIG. 2G). The small RNAs produced by incubation of dsRNAin the wheat germ extract are a mixture of ˜21-nt and 24-25-nt species.Digestion of this mixture with single-stranded nucleases produced afaster-migrating population of RNA species whose length distribution isconsistent with the original mixture having the single-strandedoverhangs and double-stranded body characteristic of siRNAs (FIG. 2G).Both size classes of small RNAs produced upon incubation of dsRNA inwheat germ extract have 2′,3′-hydroxyl and 5′ monophosphate termini(data not shown). In sum, the small RNAs have all the hallmarks of theproducts of Dicer-mediated cleavage of dsRNA. It is concluded that theyare bona fide siRNAs.

Example III: Different Dicer-Like Enzymes Produce Each Class of siRNA

There are at least two mechanisms by which long dsRNA could be convertedin plants into distinct size classes of small RNAs. Local dsRNA sequencemight determine siRNA length, irrespective of which Dicer orthologcleaves the dsRNA. In this case, we anticipate that the two classes ofsmall RNAs would have distinct sequence compositions. Instead, only the5′ ends of the two classes show sequence bias (FIG. 1B). An alternativeexplanation is that different Dicer orthologs produce each class. Boththe Arabidopsis and rice genomes encode at least four differentDicer-like proteins, including the Arabidopsis protein CARPELFACTORY/SHORT INTEGUMENTS-1 (CAF). The number of wheat Dicer orthologsis presently unknown, because the hexaploid wheat genome remains to besequenced.

Drosophila Dicer binds tightly to siRNAs (P. D. Zamore and B. Haley,unpubl.). Therefore, we reasoned that different Dicer orthologs might bedifferentially inhibited by their products, siRNAs. We tested theability of 21-nt and 25-nt synthetic siRNA duplexes to inhibit theproduction of siRNAs in Drosophila embryo lysates and the production ofthe two distinct classes of siRNA in wheat germ extract. DrosophilaDicer produces siRNAs 21-22 nt long. Drosophila Dicer was inhibited morestrongly by a 21-nt siRNA duplex than by a 25-mer (FIG. 3A). Conversely,production of 24-25-nt siRNAs by wheat germ extract was inhibited morestrongly by an ˜25-nt synthetic siRNA duplex competitor than a 21-mer(FIG. 3B). These results are consistent with the idea that the authenticsiRNA product of Dicer should bind more strongly to its active site thanan siRNA of an inauthentic length. Surprisingly, production of the˜21-nt siRNAs was completely refractory to inhibition by either 21-nt or25-nt synthetic siRNA duplexes, at siRNA concentrations as high as 800nM (FIG. 3B). The simplest explanation for these data is that adifferent Dicer-like enzyme generates each class of siRNA and that theenzyme responsible for producing the 24-25-nt siRNAs is stronglyinhibited by its siRNA product, whereas the enzyme that produces the˜21-nt siRNAs is not inhibited by siRNA product at the concentrationstested. An alternative explanation is that the concentration of theenzyme that produces the ˜21-mers is higher than the highestconcentration of inhibitor we tested, 800 nM. For this to be true, theenzyme would need to be present at micromolar concentration in theextract, which seems unlikely, as it would then correspond to ˜1% oftotal protein. The finding that production of both classes of siRNAswere equally and strongly inhibited by long dsRNA competitor (FIG. 3C)also supports an argument against this view. If the enzyme thatgenerates the 21-mers were present in the extract at very highconcentration, its activity should not have been competed by the sameconcentrations of long dsRNA competitor that saturate the enzyme thatproduces the 24-25-nt products. It is concluded that each class of siRNAis produced by the ATP-dependent, endonucleolytic cleavage of dsRNA by adifferent Dicer ortholog.

Example IV: An RNA-Dependent RNA Polymerase Activity in Wheat GermExtracts

Genetic evidence implicates an RNA-dependent RNA polymerase (RdRP) inPTGS triggered by transgenes expressing sense mRNA (S-PTGS; Dalmay etal. 2000; Mourrain et al. 2000). Plant RdRPs have been proposed togenerate dsRNA from aberrantly expressed single-stranded RNA, therebyleading to the production of siRNAs that silence that RNA (Vaucheret etal. 2001). No direct biochemical evidence has yet been presenteddemonstrating that such a pathway is plausible.

Wheat germ extracts contain an RdRP activity (FIG. 4). Increasingconcentrations of single-stranded RNA were incubated with the extractand ribonucleotide triphosphates, including [alpha]-32P-UTP.Single-stranded RNA ranging from 77 to 501 nt, either bearing a7-methyl-G(5′)ppp(5′)G or an A(5′)ppp(5′) cap structure, all led to theincorporation of 32P into RNA with approximately the same length as theexogenous, nonradioactive single-stranded RNA (FIG. 4). Theseradioactive RNAs correspond to bona fide complementary RNA (cRNA)generated by an RdRP that copied the single-stranded RNA by initiatingRNA synthesis at the extreme 3′ end of the exogenous template RNA (datanot shown). In theory, these newly radioactive RNAs could have arisen bytransfer of radiolabel to the input RNA itself. This type of labeltransfer had previously been observed when similar experiments wereperformed using Drosophila embryo lysates, but not with wheat germextract. Instead, the 32P-RNA represents newly synthesized cRNA producedby a wheat enzyme using exogenous single-stranded RNA as a template inthe absence of an exogenous nucleic acid primer

In addition to copying single-stranded RNA into approximatelyfull-length cRNA, RdRPs have also been reported to extend primers, usingsingle-stranded RNA as a template (e.g., Schiebel et al. 1998). The RdRPactivity or activities in wheat germ extract could similarly extend a32P-radiolabeled primer (FIG. 5A), but only when the RNA primer wascomplementary (antisense) to the template RNA. Under identicalconditions, no such primer-extension activity was detected in lysates ofsyncitial blastoderm Drosophila embryos, despite earlier reports to thecontrary (Lipardi et al. 2001). RNA-dependent, RNA-primer-extensionactivity was detected, however, when wheat and fly extracts are mixed(FIG. 5A). In neither Drosophila embryo lysate nor wheat germ extractcan we detect primer extension of a single-stranded RNA template using a21-nt siRNA duplex rather than a 21-nt antisense primer.

It is proposed that aberrant single-stranded RNA triggers silencing inplants when it serves as a template for the production of cRNA,generating dsRNA, which can then be cleaved by Dicer into siRNAduplexes. These data suggest that such copying does not require primers,but is triggered merely by an exceptionally high concentration ofsingle-stranded RNA. To test if high concentrations of single-strandedmRNA could lead to the production of siRNAs, the RdRP reactions wererepeated using a 2.7-kb single-stranded firefly luciferase mRNA.Increasing concentrations of the mRNA were incubated in either wheatgerm extract or Drosophila embryo lysate in the presence of ATP, CTP,GTP, and [alpha]-32P-UTP, and examined for the production of 21-25-ntradioactive RNAs. FIG. 5B (left) shows that when the incubations wereperformed in wheat germ lysates, a single class of small RNA, ˜24 ntlong, was produced with increasing concentrations of the exogenous,single-stranded template RNA. No such radioactive product was observedin Drosophila embryo lysates, but it is noted that these lysates containendogenous UTP, which may preclude detection of 32P small RNAs. To testif the radiolabeled ˜24-nt products were generated by the de novosynthesis of RNA, the experiment was repeated, replacing CTP and GTPwith 3′-deoxy CTP and 3′-deoxy GTP, inhibitors of RNA synthesis. In thepresence of these inhibitors, no radioactive small RNAs were observed inthe wheat reaction (FIG. 5B, right). Thus, single-stranded RNA cantrigger in wheat germ extract the de novo synthesis of ˜24-nt smallRNAs.

Notably, the production of 21-nt RNAs was not detected in this assay.The assay should have detected such 21-nt small RNAs if they werepresent at 1/10 the concentration of the ˜24-mers, but we would beunlikely to detect them far below this threshold. Experiments withdouble-stranded RNA suggest that the 21-mers are produced in wheat atabout ¼ the rate of the 24-25-nt small RNAs (FIG. 2). Thus, theproduction of dsRNA by the RdRP activity may be coupled to theproduction of the longer class of small RNAs. It is noted that suchcoupling does not imply that production of ˜24-nt siRNAs from exogenousdsRNA requires the participation of an RdRP. It is proposed that dsRNAgenerated by RdRP copying of single-stranded RNA is preferentiallyprocessed by a wheat Dicer ortholog that produces long siRNAs, perhapsbecause the two proteins are physically linked.

The question of whether the ˜24-nt RNAs synthesized in the RdRPreactions are actual products of Dicer cleavage of dsRNA was nextaddressed. Production of wheat 24-25-nt siRNAs from 32P-radiolabeleddsRNA is efficiently inhibited by synthetic siRNA duplexes; 25-ntsynthetic siRNA duplexes are more potent inhibitors than 21-nt duplexes(FIG. 3B). Therefore, an experiment was conducted to determine ifproduction of the ˜24-nt small RNAs in the RdRP reactions was similarlyinhibited by synthetic siRNA duplexes. FIG. 5C shows that the productionof ˜24-nt small RNAs in the RdRP reactions programmed with a 2.7-kbsingle-stranded RNA template was inhibited by synthetic siRNA duplexes.Like the production of 24-25-nt siRNAs from exogenous dsRNA, productionof the de novo synthesized ˜24-mers was inhibited to a greater extent by25-nt synthetic siRNA duplexes than by 21-nt duplexes (FIG. 5C).Half-maximal inhibition of small RNA production in the RdRP-dependentreactions occurred at roughly the same concentration of synthetic siRNAduplex as inhibition of the processing of 32P dsRNA (cf. FIGS. 5C and3B). It is concluded that in wheat germ extract, exogenoussingle-stranded RNA provides the template for the synthesis of cRNA byan RdRP and that the resulting template-RNA:cRNA hybrid is thenpreferentially cleaved into ˜24-nt siRNAs by a Dicer-like enzyme.

Example V: miRNAs Act as siRNAs in Plants

In addition to siRNAs, another class of small RNAs, microRNAs (miRNAs),has been detected in plants (Llave et al. 2002a; Park et al. 2002;Reinhart et al. 2002). Like their animal counterparts, plant miRNAs aregenerated by a Dicer family member, CAF. miRNAs are encoded in stem-loopprecursor RNAs that are cleaved by CAF into 21-24-nt single-strandedsmall RNAs (Park et al. 2002; Reinhart et al. 2002). Exogenous miRNAprecursors were not faithfully processed into mature miRNAs in wheatgerm extract (data not shown). Instead, in vitro transcribed pre-miRNAswere cleaved into small RNAs too long to correspond to authentic, maturemiRNAs. Perhaps the Dicer ortholog responsible for miRNA maturation inwheat—presumably wheat CAF—is absent from wheat germ extracts. InArabidopsis, CAF transcripts that encode a protein with a nuclearlocalization signal have been reported, suggesting that CAF protein maybe nuclear (Jacobsen et al. 1999). Because wheat germ extracts areessentially cytoplasm, nuclear CAF might not be present in the extract.

Plant miRNAs differ from animal miRNAs in that there are correspondingmRNA sequences in the Arabidopsis and rice genomes with significantcomplementarity to miRNA sequences (Llave et al. 2002a,b; Reinhart etal. 2002; Rhoades et al. 2002). The high degree of complementaritybetween 14 recently analyzed plant miRNAs and specific families ofdevelopmentally important plant mRNAs led to the proposal that plantmiRNAs direct developmentally controlled mRNA destruction (Rhoades etal. 2002). That is, after the plant miRNAs are generated by the cleavageof pre-miRNAs by CAF, they enter the RNAi pathway and function assiRNAs. In contrast, animal miRNAs are thought to act as translationalrepressors (for review, see Ruvkun 2001). An untested feature of thisproposal is that an RNAi-like pathway in plants tolerates the three tofour mismatches sometimes observed between an miRNA and its predictedmRNA target.

If plant miRNAs are endogenous mediators of RNAi, then wheat germextracts should contain miRNA-programmed complexes that specifyendonucleolytic cleavage of corresponding target RNAs. In particular,miR165 has been proposed to down-regulate PHV and PHABULOSA (PHB) mRNAexpression in Arabidopsis by an RNAi-like mechanism (Rhoades et al.2002). PHV and PHB encode homeodomain-leucine zipper transcriptionfactors implicated in the perception of radial position in the shoottissues that give rise to leaves (McConnell and Barton 1998; McConnellet al. 2001). Dominant phv and phb mutations alter a single amino acid(glycine→glutamic acid) in the sterol/lipid-binding domain of theproteins, suggesting that the mutant phenotype results from a change inthe function of PHV and PHB (McConnell and Barton 1998; McConnell et al.2001). However, the discovery of plant miRNAs complementary to this sitein PHV led to the suggestion that the molecular basis of the dominanceis the persistence of PHV and PHB expression at developmental stageswhen these mRNAs are normally destroyed (Rhoades et al. 2002). Thishypothesis is consistent with both the increased overall levels of PHBmRNA in the dominant mutant and the increased activity of a dominantmutant phb mRNA on the abaxial, rather than the adaxial, domain of theleaf primordium (McConnell and Barton 1998; McConnell et al. 2001).

miR165 or miR166 is present in wheat germ extracts (FIG. 6A). miR165 andmiR166 differ by a single C-to-U transition that decreases thecomplementarity of miR166 to PHV and PHB by changing a G:C base pair toa G:U wobble. Rice (Oryza) is the sequenced genome most closely relatedto wheat. Although the rice genome encodes no miR165 homolog, it encodessix copies of miR166 (Reinhart et al. 2002). Because the Northernhybridization conditions used herein cannot distinguish between miR165and miR166, the endogenous wheat miRNA is referred to as miR165/166.

To begin to test the hypothesis that plant miRNAs function to regulatetarget gene expression by an RNAi-like mechanism, target RNAs wereprepared encoding a portion of the wild-type sequence of Arabidopsis PHVor the dominant G→A point mutation, which falls within the PHV sequencesproposed to pair with miR165/166. The target RNAs and relevant miRNAsare shown in FIG. 6C. 5′-radiolabeled target RNAs were incubated withwheat germ extract, then analyzed on a denaturing sequencing gel. In theabsence of any other exogenous RNA, the wild-type PHV target RNA, butnot the dominant G→A mutant, was efficiently cleaved within the regioncomplementary to miR165/166 (FIG. 7A,B). This 21-nt region is identicalin PHV and PHB, and a target RNA that contained sequence from theArabidopsis PHB mRNA was also cleaved within the sequences complementaryto miR165/166 upon incubation in the wheat germ extract (data notshown). In the RNAi pathway, a key feature of small RNA-directed targetdestruction is that pretreatment with the single-stranded nucleicacid-specific enzyme, micrococcal nuclease, abolishes RISC activity(Hammond et al. 2000). Cleavage of the PHV target RNA was likewiseabolished by pretreatment of the extract with micrococcal nuclease (datanot shown), consistent with the view that miR165/166 acts as a guide todirect target cleavage. The difference in cleavage rate betweenwild-type and mutant target RNAs, which differ only at a singlenucleotide, was >14-fold (FIG. 7B). Thus, the resistance of the mutantphv RNA to cleavage by an endogenous RNAi-like nuclease can explain whythe mutation is dominant.

Next, cleavage of the PHV target RNA by various siRNAs was analuzed inDrosophila embryo lysate (FIG. 6C). An siRNA with perfectcomplementarity to the site predicted to pair with miR165/166 and ansiRNA duplex in which one strand had the sequence of miR165 or miR166directed cleavage of the PHV target RNA, yielding the predicted 514-nt5′ cleavage product (FIG. 7C). None of these three siRNAs efficientlycleaved the PHV mutant target (FIG. 7C). Quantification of the cleavagemediated by the siRNA with perfect complementarity as compared to thatmediated by miR165 demonstrates that miR165 more efficiently mediatestarget cleavage. This is presumed to be due to the fact that miR 165forms two G:U wobble base pairs with the target mRNA, one at position 1and one at position 17 (with respect to the 5′ end of the antisensesiRNA strand) (FIG. 8).

The failure of the miR165-siRNA duplex to cleave mutant PHV was a directconsequence of its reduced complementarity to the target RNA at position6 (with respect to the 5′ end of the antisense siRNA strand), because ansiRNA with perfect complementarity to the mutant sequence (FIG. 6B)efficiently cleaved the mutant RNA (FIG. 7C). The 5′ cleavage productproduced in the siRNA-programmed RNAi reactions comigrated with thatproduced when the PHV target RNA was incubated in wheat germ extractwithout exogenous siRNA (FIG. 7C).

The simplest explanation for the sequence-specificity of the nuclease isthat it is guided by miR165/166: cleavage requires a nucleic acidcomponent, occurs at the same site on the PHV target RNA as directed byan siRNA duplex with the sequence of miR165 or miR166 in Drosophilaembryo lysate, and, like the siRNA, is inefficient with the G→A mutantphv RNA. In the RNAi pathway, an siRNA-programmed endonuclease complexis called an RISC (Hammond et al. 2000). These data suggest that wheatmiR165/166 is in an RISC, supporting the proposal that plant miRNAsregulate expression of their mRNA targets by endogenous RNAi.

Example VI: miR165/166 Directs Multiple Rounds of Target Cleavage

The next question addressed was whether the miR165/166-programmed RISCacts as an enzyme. Quantitative Northern hybridization demonstrates thatthe wheat germ extract reactions contained 0.083 nM miR165/166 (FIG.6B). The target RNA concentration in these reactions was 5 nM, and morethan half the target RNA was destroyed in 80 min (FIG. 7A). Thus, eachmiR165/166 RNA directed cleavage of ˜30 target RNA molecules. Therefore,the miR165/166-programmed RISC is a multiple-turnover enzyme.

Discussion

The above data show that wheat germ extracts recapitulate in vitro manyaspects of RNA silencing in plants. Wheat germ extracts convertexogenous dsRNA into two distinct classes of small RNAs. Detailedanalysis of these small RNAs indicates that they are bona fide siRNAs.Thus, plant siRNAs are derived directly from longer dsRNA, just as inanimals. The data indicate that distinct Dicer-like enzymes generate thetwo functionally distinct classes of siRNAs. Cloned endogenous smallRNAs from Arabidopsis likewise form two distinct length classes, whose5′ ends indicate that they are made by distinct enzymes. An alternativeview, that one or more Dicer-like enzymes may generate both classes ofsmall RNAs, with the different lengths a byproduct of local sequencecontext, is not consistent with the above observation that production of24-25-nt RNAs in wheat germ extract was inhibited by synthetic siRNAduplexes, whereas ˜21-nt siRNA production was not. If the production ofsiRNAs is tightly coupled to the assembly of downstream effectorcomplexes, then their production by different Dicer orthologs may ensurethat the two classes of siRNAs function in different cellular pathways(see also Hamilton et al. 2002).

A hallmark of PTGS in plants and RNAi in nematodes is the spreading ofsilencing signals along the length of the mRNA target. In plants,spreading occurs in both the 5′ and 3′ directions and requires theputative RdRP gene, SGS2. Spreading is observed even when silencing isinitiated by a single siRNA sequence (Klahre et al. 2002). Onehypothesis is that 5′ spreading is initiated by the antisense siRNAstrand priming copying of the target mRNA by an RdRP, thereby producingdsRNA. 3′ spreading cannot be explained by such a mechanism. Both 5′ and3′ spreading might instead be catalyzed by the conversion of mRNAfragments into dsRNA by an RdRP that initiates synthesis at the 3′ endof the two fragments generated when an RISC cleaves the target RNA. ThisdsRNA would then be cleaved by a Dicer-like enzyme to produce secondarysiRNAs (Lipardi et al. 2001; Sijen et al. 2001). Such RNA synthesiswould occur without the involvement of a primer. The above datademonstrate that exogenous single-stranded RNA is copied into cRNA inthe extract by a wheat RdRP that acts without the aid of an exogenousprimer. The resulting dsRNA is cleaved preferentially into the longerclass of siRNAs, suggesting the RdRP is physically linked to a specificDicer ortholog. The specific biochemical function of the 24-25-nt siRNAsgenerated in this reaction remains to be determined.

miRNAs Function as siRNAs in Plants

The above data further show that miRNAs in plants function in much thesame way that siRNA duplexes function in Drosophila and humans: asguides for an endonuclease complex. Each endonuclease complex cancatalyze multiple rounds of target cleavage, indicating that the miRNAis not consumed in the reaction. Entry of a miRNA into amultiple-turnover RNAi enzyme complex is not unprecedented; in humancells, the miRNA let-7 is a component of an RISC, although the humangenome does not appear to contain any mRNA sequences with sufficientcomplementarity to be cleaved by this RISC (Hutvágner and Zamore 2002).Like the plant miR165/166-programmed RISC, the human let-7-programmedRISC can catalyze multiple rounds of target cleavage.

Additional support for the idea that plant miRNAs direct cleavage ofcomplementary mRNA targets comes from the work of Carrington andcolleagues, who recently showed that a family of Arabidopsis mRNAsencoding SCARECROW-LIKE (SCL) transcription factors is cleaved by anRNAi-like process directed by miR171, an miRNA that is fullycomplementary to its mRNA targets, unlike miR165/166 (Llave et al.2002b). Like wheat miR165/166, Arabidopsis miR171 appears to direct theendonucleolytic cleavage of its target mRNAs. In this respect, miR171functions as if it were a single-stranded siRNA. Single-stranded siRNAscan trigger RNAi in both Drosophila and mammalian cell extracts and invivo in HeLa cells (Martinez et al. 2002a; Schwarz et al. 2002),although much higher concentrations of single-stranded siRNA is requiredthan for duplex (Schwarz et al. 2002). Furthermore, an individual humanRISC contains only one strand of the exogenous siRNA duplex used totrigger RNAi (Martinez et al. 2002a).

The observation that, in Drosophila embryo lysate, an siRNA with thesequence of miR165, which contains three mismatches with its targetmRNA, is at least as potent as an siRNA with perfect complementarity tothe same target sequence, demonstrates that mismatches per se do notblock target cleavage. Rather, the specific position and sequence ofsiRNA:target RNA mismatches determine if they permit or disrupt RNAi.The data also suggest that miRNAs in plants evolved to optimize cleavageefficiency rather than maximize complementarity to their targets. It ispredicted that three or four mismatches between an miRNA (or the guidestrand of an siRNA duplex) and its target RNA, properly placed so as tostill permit mRNA cleavage, will facilitate the release of cleavedtarget RNA from the RISC complex, thereby increasing the rate of enzymeturnover.

miRNA Function and the Spread of Silencing Signals Along a SilencedSequence

Spreading of silencing signals along the length of a silenced mRNAsequence is a common feature of plant RNA silencing. Because miRNAs actas siRNAs, one might anticipate that they would also elicit spreading.However, miRNA-induced spreading is not consistent with the genetics ofthe PHV and PHB mutants; the very existence of a dominant PHV mutantexcludes both 5′ and 3′ spreading. Spreading of the silencingsignal—that is, the generation of new siRNAs 5′ or 3′ to the site ofinitial target cleavage—would produce siRNAs containing sequences commonto both the wild-type and mutant PHV mRNAs. If such siRNAs weregenerated, they would direct destruction of the mutant PHV mRNA. In sucha case, the PHV mutant could only have been recovered as a recessive,not a dominant allele. Genetic studies (McConnell et al. 2001) show thatendonucleolytic cleavage of target RNAs by miRNA-directed RISC complexesdoes not trigger spreading in plants. This remains true even when themiRNA is the perfect complement of its mRNA target (Llave et al. 2002b).

How, then, can the well-documented spreading phenomenon observed forS-PTGS be reconciled with the absence of spreading in miRNA-directedtarget cleavage? It is proposed that plants contain two separatemechanisms for target mRNA destruction—endogenous mRNAs are regulated byendonucleolytic cleavage directed by miRNA-programmed RISC complexes,whereas exogenous silencing triggers, such as transgenes or viruses,might initiate successive cycles of siRNA-primed, RdRP-catalyzed dsRNAsynthesis, followed by cleavage of the dsRNA into siRNAs by Dicer-likeenzymes, a mechanism termed random degradative PCR (Lipardi et al.2001). RISC complexes would play no role in the execution of target RNAsin this cycle. The observation that a single siRNA sequence can trigger3′ spreading (Klahre et al. 2002) is difficult to reconcile with apriming mechanism. Intriguingly, VIGS-mediated RNA silencing ofendogenous genes is not associated with spreading of silencing intoregions of the target sequence 5′ or 3′ to the initial silencing trigger(Vaistij et al. 2002), although such silencing clearly must involvesiRNAs derived from viral dsRNA, not endogenous miRNAs.

An alternative hypothesis is that the absolute concentration of an RNAtarget might determine if the 5′ and 3′ cleavage fragments generated bytarget cleavage are converted into dsRNA by an RdRP. Only when theproducts of RISC-mediated target cleavage accumulate to a sufficientlyhigh concentration would they serve as substrates for the RdRP andconsequently trigger spreading. Experiments withpolygalacturonase-silenced tomatoes support this view (Han and Grierson2002). In these plants, siRNAs were produced from the silencing-inducingtransgene but not the corresponding silenced endogene. The siRNAs werepreferentially produced from the 3′ end of the transgene, consistentwith the idea that plant RdRPs act without aid of a primer. Furthermore,these authors detected mRNA degradation products consistent withendonucleolytic cleavage of the targeted polygalacturonase endogene.Thus, RISC-mediated cleavage per se does not appear to trigger spreadingalong the target RNA sequence. More likely, the endonucleolytic cleavageof transgenic mRNA produces a sufficiently high concentration of mRNAfragments to recruit an RdRP, resulting in the production of siRNAs fromthe 3′ cleavage product. miRNA-directed cleavage of natural plantregulatory targets would not lead to spreading, because endogenous mRNAtargets are not present at sufficiently high concentrations to recruitthe RdRP. This model predicts that the putative RdRP SGS2 (SDE1)required for PTGS, will not be required for miRNA-directed destructionof endogenous mRNA targets. In fact, no developmental abnormalities havebeen reported for SGS2 mutants (Mourrain et al. 2000), includingmutations likely to be strongly hypomorphic or functionally null (Dalmayet al. 2000), suggesting that plants lacking SGS2 protein have normalmiRNA biogenesis and function.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-44. (canceled)
 45. A vector encoding an engineered pre-miRNAcomprising an RNAi agent which comprises a sense strand and an antisensestrand; wherein the antisense strand is sufficiently complementary to atarget sequence in a wild-type human mRNA; and wherein one or moreterminal nucleotides that are within 5 or fewer nucleotides from the 5′end of the antisense strand are substituted with nucleotides which formG:U wobble base pairs with the corresponding nucleotides in the targetsequence when compared with a reference antisense strand that is fullycomplementary to the target sequence.
 46. The vector of claim 45,wherein the vector is an adeno-associated virus (AAV) vector.
 47. Thevector of claim 46, wherein the antisense strand is 19-22 nucleotides inlength.
 48. The vector of claim 47, wherein only 1-3 of the one or moreterminal nucleotides are substituted with nucleotides which form G:Uwobble base pairs.
 49. The vector of claim 47, wherein only 1-2 of theone or more terminal nucleotides are substituted with nucleotides whichform G:U wobble base pairs.
 50. The vector of claim 47, wherein only 1-2terminal nucleotides that are within 3 or fewer nucleotides from the 5′end of the antisense strand are substituted with nucleotides which formG:U wobble base pairs; wherein the last two nucleotides at the 3′ end ofthe antisense strand are UU; and wherein all nucleotides in theantisense strand are perfectly complementary to the target sequenceother than the UU nucleotides at the 3′ end of the antisense strand andthe 1-2 terminal nucleotides which are substituted to form a G:U wobblebase pair.
 51. The vector of claim 47, wherein only one of the one ormore terminal nucleotides is substituted with a nucleotide which forms aG:U wobble base pair.
 52. The vector of claim 47, wherein only oneterminal nucleotide that is within 3 or fewer nucleotides from the 5′end of the antisense strand is substituted with a nucleotide which formsa G:U wobble base pair; wherein the last two nucleotides at the 3′ endof the antisense strand are UU; and wherein all nucleotides in theantisense strand are perfectly complementary to the target sequenceother than the UU nucleotides at the 3′ end of the antisense strand andthe one terminal nucleotide which is substituted to form a G:U wobblebase pair.
 53. The vector of claim 47, wherein the first terminalnucleotide at the 5′ end of the antisense strand is substituted with anucleotide which forms a G:U wobble base pair; wherein the last twonucleotides at the 3′ end of the antisense strand are UU; and whereinall nucleotides in the antisense strand are perfectly complementary tothe target sequence other than the UU nucleotides at the 3′ end of theantisense strand and the one terminal nucleotide which is substituted toform a G:U wobble base pair.
 54. The vector of claim 53, wherein thesubstitution is a C→U substitution, with the U forming a G:U wobble basepair with a corresponding G in the target sequence in the wild-typehuman mRNA.
 55. A pharmaceutical composition comprising the vector ofclaim
 50. 56. The vector of claim 50, wherein the vector encodes apri-miRNA which comprises the pre-miRNA.
 57. A vector encoding anengineered pre-miRNA, said pre-miRNA comprising a single strand thatforms a hairpin which comprises a duplex stem and loop; said duplex stemcomprising an RNAi agent formed by a sense strand on a first portion ofthe stem and an antisense strand on a second portion of the stem;wherein each of the first and second stem portions are 18-25 nucleotidesin length and the loop is 2-9 nucleotides in length; wherein theantisense strand is sufficiently complementary to a target sequence in awild-type human mRNA; and wherein one or more terminal nucleotides thatare within 5 or fewer nucleotides from the 5′ end of the antisensestrand are substituted with nucleotides which form G:U wobble base pairswith the corresponding nucleotides in the target sequence when comparedwith a reference antisense strand that is fully complementary to thetarget sequence.
 58. The vector of claim 57, wherein the vector is anadeno-associated virus (AAV) vector.
 59. The vector of claim 58, whereinthe antisense strand is 19-22 nucleotides in length.
 60. The vector ofclaim 59, wherein only 1-3 of the one or more terminal nucleotides aresubstituted with nucleotides which form G:U wobble base pairs.
 61. Thevector of claim 59, wherein only 1-2 of the one or more terminalnucleotides are substituted with nucleotides which form G:U wobble basepairs.
 62. The vector of claim 59, wherein only 1-2 terminal nucleotidesthat are within 3 or fewer nucleotides from the 5′ end of the antisensestrand are substituted with nucleotides which form G:U wobble basepairs; wherein the last two nucleotides at the 3′ end of the antisensestrand are UU; and wherein all nucleotides in the antisense strand areperfectly complementary to the target sequence other than the UUnucleotides at the 3′ end of the antisense strand and the 1-2 terminalnucleotides which are substituted to form a G:U wobble base pair. 63.The vector of claim 59, wherein only one of the one or more terminalnucleotides is substituted with a nucleotide which forms a G:U wobblebase pair.
 64. The vector of claim 59, wherein only one terminalnucleotide that is within 3 or fewer nucleotides from the 5′ end of theantisense strand is substituted with a nucleotide which forms a G:Uwobble base pair; wherein the last two nucleotides at the 3′ end of theantisense strand are UU; and wherein all nucleotides in the antisensestrand are perfectly complementary to the target sequence other than theUU nucleotides at the 3′ end of the antisense strand and the oneterminal nucleotide which is substituted to form a G:U wobble base pair.65. The vector of claim 59, wherein the first terminal nucleotide at the5′ end of the antisense strand is substituted with a nucleotide whichforms a G:U wobble base pair; wherein the last two nucleotides at the 3′end of the antisense strand are UU; and wherein all nucleotides in theantisense strand are perfectly complementary to the target sequenceother than the UU nucleotides at the 3′ end of the antisense strand andthe one terminal nucleotide which is substituted to form a G:U wobblebase pair.
 66. The vector of claim 65, wherein the substitution is a C→Usubstitution, with the U forming a G:U wobble base pair with acorresponding G in the target mRNA sequence.
 67. A pharmaceuticalcomposition comprising the vector of claim
 62. 68. The vector of claim62, wherein the vector encodes a pri-miRNA which comprises thepre-miRNA.